bluuebunny/crossref_metadata_2025 huggingface dataset:
Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli
Authors: Chiorini J A , Weitzman M D , Owens R A , Urcelay E , Safer B , Kotin R M
Published: January 2020

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.
Antigenic determinants of measles virus hemagglutinin associated with neurovirulence
Authors: Liebert U G , Flanagan S G , Löffler S , Baczko K , ter Meulen V , Rima B K
Published: January 2020

The biological activity of monoclonal antibodies specific for the hemagglutinin protein of measles virus strain CAM recognizing six epitope groups according to their binding properties to measles virus strain CAM/R401 was investigated in vivo in our rat model of measles encephalitis. When injected intraperitoneally into measles virus-infected suckling rats, some monoclonal antibodies modified the disease process and prevented the necrotizing encephalopathy seen in untreated animals. The analysis of measles virus brain isolates revealed emergence of variants that resisted neutralization with the passively transferred selecting monoclonal antibody but not with other monoclonal antibodies. Monoclonal antibody escape mutants were also isolated in vitro, and their neurovirulence varied in the animal model. Sequence data from the hemagglutinin gene of measles virus localize a major antigenic surface determinant of the hemagglutinin protein between amino acid residues 368 and 396, which may be functionally important for neurovirulence. The data indicate that the interaction of antibodies with the measles virus H protein plays an important role in the selection of neurovirulent variants. These variants have biological properties different from those of the parent CAM virus.
pet, a small sequence distal to the pregenome cap site, is required for expression of the duck hepatitis B virus pregenome
Authors: Huang M , Summers J
Published: January 2020

We have found that transcription of the pregenome of an avian hepadnavirus, duck hepatitis B virus (DHBV), is dependent on the presence of a small element in the 5' transcribed region of the pregenome-encoding sequence. This element, which we have named pet (positive effector of transcription), exerts its effect in cis in a position and orientation-dependent manner, suggesting that it may function as part of the nascent pregenome transcript. The requirement for pet depends on the presence in the transcription unit of a region of the DHBV genome located upstream of the envelope promoters, which specifically suppresses transcription of templates lacking pet. In the presence of this region, deletion of pet activates transcription from downstream promoters, suggesting that pregenome transcription complexes fail to reach the downstream promoters. In vitro transcription experiments support the model that pet is required for transcription elongation on the DHBV template. We speculate that pet is required to suppress transcription termination during the first passage of pregenome transcription complexes through a viral termination region on the circular viral DNA.
Hormonal regulation of the gene for the type C ecotropic retrovirus receptor in rat liver cells
Authors: Wu J Y , Robinson D , Kung H J , Hatzoglou M
Published: January 2020

The infectibility of the regenerating rat liver by ecotropic retroviruses was studied relative to the expression of the gene coding for the ecotropic retrovirus receptor (Ecor) that functions as a cationic amino acid transporter. It is known that the gene for the receptor is expressed in primary hepatocytes and hepatoma cells but is absent in adult liver cells. Isolation of a 2.85-kb cDNA for the rat Ecor suggested that the rat viral receptor is 97% homologous to the mouse viral receptor and that it contains the envelope-binding domain that determines the host range of ecotropic murine retroviruses. This explains the efficient infection of rat cells by ecotropic retroviruses. Since cell division is required for liver cells to be infected, we determined the susceptibility of the regenerating rat liver to infection at different time points after partial hepatectomy (0 to 24 h) in relation to the presence of receptor mRNA. Infection of the liver occurred only when the liver was exposed to virus 4 h after partial hepatectomy. This time course of infection paralleled expression of the gene for the Ecor, which was rapidly induced between 2 and 6 h during liver regeneration. However, expression of the dormant receptor gene in quiescent liver cells can be induced by insulin, dexamethasone, and arginine, indicating that cell division is not required for expression of the receptor gene in liver cells. A diet high in carbohydrate (low in protein) significantly increased the concentration of receptor mRNA in liver cells, indicating that hormones play a role in the regulation of expression of this gene in vivo. We conclude that the gene for the viral receptor is expressed in the regenerating and quiescent liver when the urea cycle enzymes are down regulated. The infection of the regenerating rat liver by ecotropic retroviruses at the time point of expression of the receptor gene supports the requirement of expression of this transporter for infection.
Mucosal model of genital immunization in male rhesus macaques with a recombinant simian immunodeficiency virus p27 antigen
Authors: Lehner T , Tao L , Panagiotidi C , Klavinskis L S , Brookes R , Hussain L , Meyers N , Adams S E , Gearing A J , Bergmeier L A
Published: January 2020

Human immunodeficiency virus (HIV) can be transmitted through infected seminal fluid or vaginal or rectal secretions during heterosexual or homosexual intercourse. To prevent mucosal transmission and spread to the regional lymph nodes, an effective vaccine may need to stimulate immune responses at the genitourinary mucosa. In this study, we have developed a mucosal model of genital immunization in male rhesus macaques, by topical urethral immunization with recombinant simian immunodeficiency virus p27gag, expressed as a hybrid Ty virus-like particle (Ty-VLP) and covalently linked to cholera toxin B subunit. This treatment was augmented by oral immunization with the same vaccine but with added killed cholera vibrios. Polymeric secretory immunoglobulin A (sIgA) and IgG antibodies to p27 were induced in urethral secretions, urine, and seminal fluid. This raises the possibility that the antibodies may function as a primary mucosal defense barrier against SIV (HIV) infection. The regional lymph nodes which constitute the genital-associated lymphoid tissue contained p27-specific CD4+ proliferative and helper T cells for antibody synthesis by B cells, which may function as a secondary immune barrier to infection. Blood and splenic lymphocytes also showed p27-sensitized CD4+ T cells and B cells in addition to serum IgG and IgA p27-specific antibodies; this constitutes a third level of immunity against dissemination of the virus. A comparison of genito-oral with recto-oral and intramuscular routes of immunization suggests that only genito-oral immunization elicits specific sIgA and IgG antibodies in the urine, urethra, and seminal fluid. Both genito-oral and recto-oral immunizations induced T-cell and B-cell immune responses in regional lymph nodes, with preferential IgA antibody synthesis. The mucosal route of immunization may prevent not only virus transmission through the genital mucosa but also dissemination and latency of the virus in the draining lymph nodes.
Proteolytic cleavage of bovine herpesvirus 1 (BHV-1) glycoprotein gB is not necessary for its function in BHV-1 or pseudorabies virus
Authors: Kopp A , Blewett E , Misra V , Mettenleiter T C
Published: January 2020

Glycoprotein B homologs represent the most highly conserved group of herpesvirus glycoproteins. They exist in oligomeric forms based on a dimeric structure. Despite the high degree of sequence and structural conservation, differences in posttranslational processing are observed. Whereas gB of herpes simplex virus is not proteolytically processed after oligomerization, most other gB homologs are cleaved by a cellular protease into subunits that remain linked via disulfide bonds. Proteolytic cleavage is common for activation of viral fusion proteins, and it has been shown that herpesvirus gB homologs are essential for membrane fusion events during infection, e.g., virus penetration and direct viral cell-to-cell spread. To analyze the importance of proteolytic cleavage for the function of gB homologs, we isolated a mutant bovine herpesvirus 1 (BHV-1) expressing a BHV-1 gB that is no longer proteolytically processed because of a deletion of the proteolytic cleavage site and analyzed its phenotype in cell culture. We showed previously that BHV-1 gB can functionally substitute for the homologous glycoprotein in pseudorabies virus (PrV), based on the isolation of a PrV gB-negative PrV recombinant that expresses BHV-1 gB (A. Kopp and T. C. Mettenleiter, J. Virol, 66:2754-2762, 1992). Therefore, we also isolated a mutant PrV lacking PrV gB but expressing a noncleavable BHV-1 gB. Our results show that cleavage of BHV-1 gB is not essential for its function in either a BHV-1 or a PrV background. Compared with the PrV recombinant expressing cleavable BHV-1 gB, deletion of the cleavage site in the recombinant PrV did not detectably alter the viral phenotype, as analyzed by plaque assays, one-step growth kinetics, and penetration kinetics. In the BHV-1 mutant, the uncleaved BHV-1 gB was functionally equivalent to the wild-type protein with regard to penetration and showed only slightly delayed one-step growth kinetics compared with parental wild-type BHV-1. However, the resulting plaques were significantly smaller, indicating a role for proteolytic cleavage of BHV-1 gB in cell-to-cell spread of BHV-1.
Killer toxin-secreting double-stranded RNA mycoviruses in the yeasts Hanseniaspora uvarum and Zygosaccharomyces bailii
Authors: Schmitt M J , Neuhausen F
Published: January 2020

Killer toxin-secreting strains of the yeasts Hanseniaspora uvarum and Zygosaccharomyces bailii were shown to contain linear double-stranded RNAs (dsRNAs) that persist within the cytoplasm of the infected host cell as encapsidated virus-like particles. In both yeasts, L- and M-dsRNAs were associated with 85-kDa major capsid protein, whereas the additional Z-dsRNA (2.8 kb), present only in the wild-type Z. bailii killer strain, was capsid protein, whereas the additional Z-dsRNA (2.8 kb), present only in the wild-type Z. bailii killer strain, was shown to be encapsidated by a 35-kDa coat protein. Although Northern (RNA) blot hybridizations indicated that L-dsRNA from Z. bailii is a LA species, additional peptide maps of the purified 85-kDa capsid from Z. bailii and the 88- and 80-kDa major coat proteins from K1 and K28 killer viruses of Saccharomyces cerevisiae revealed distinctly different patterns of peptides. Electron microscopy of purified Z. bailii viruses (ZbV) identified icosahedral particles 40 nm in diameter which were undistinguishable from the S. cerevisiae killer viruses. We demonstrated that purified ZbVs are sufficient to confer the Z. bailii killer phenotype on transfected spheroplasts of a S. cerevisiae nonkiller strain and that the resulting transfectants secreted even more killer toxin that the original ZbV donor strain did. Curing experiments with ZbV-transfected S. cerevisiae strains indicated that the M-dsRNA satellite from Z. bailii contains the genetic information for toxin production, whereas expression of toxin immunity might be dependent on Z-dsRNA, which resembles a new dsRNA replicon in yeasts that is not dependent on an LA helper virus to be stably maintained and replicated within the cell.
Analysis of the signals for polarized transport of influenza virus (A/WSN/33) neuraminidase and human transferrin receptor, type II transmembrane proteins
Authors: Kundu A , Nayak D P
Published: January 2020

In polarized MDCK cells influenza virus (A/WSN/33) neuraminidase (NA) and human transferrin receptor (TR), type II glycoproteins, when expressed from cloned cDNAs, were transported and accumulated preferentially on the apical and basolateral surfaces, respectively. We have investigated the signals for polarized sorting by constructing chimeras between NA and TR and by making deletion mutants. NATR delta 90, which contains the cytoplasmic tail and transmembrane domain of NA and the ectodomain of TR, was found to be localized predominantly on the apical membrane, whereas TRNA delta 35, containing the cytoplasmic and transmembrane domains of TR and the ectodomain of NA, was expressed preferentially on the basolateral membrane. TR delta 57, a TR deletion mutant lacking 57 amino acids in the TR cytoplasmic tail, did not exhibit any polarized expression and was present on both apical and basolateral surfaces, whereas a deletion mutant (NA delta 28-35) lacking amino acid residues from 28 to 35 in the transmembrane domain of NA resulted in secretion of the NA ectodomain predominantly from the apical side. These results taken together indicate that the cytoplasmic tail of TR was sufficient for basolateral transport, but influenza virus NA possesses two sorting signals, one in the cytoplasmic or transmembrane domain and the other within the ectodomain, both of which are independently able to transport the protein to the apical plasma membrane.
Competitive quantitative PCR analysis of herpes simplex virus type 1 DNA and latency-associated transcript RNA in latently infected cells of the rat brain
Authors: Ramakrishnan R , Fink D J , Jiang G , Desai P , Glorioso J C , Levine M
Published: January 2020

Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.
Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles
Authors: Lavallée C , Yao X J , Ladha A , Göttlinger H , Haseltine W A , Cohen E A
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) particles consists of two molecules of genomic RNA as well as molecules originating from gag, pol, and env products, all synthesized as precursor proteins. The 96-amino-acid Vpr protein, the only virion-associated HIV-1 regulatory protein, is not part of the virus polyprotein precursors, and its incorporation into virus particles must occur by way of an interaction with a component normally found in virions. To investigate the mechanism of incorporation of Vpr into the HIV-1 virion, Vpr- proviral DNA constructs harboring mutations or deletions in specific virion-associated gene products were cotransfected with Vpr expressor plasmids in COS cells. Virus released from the transfected cells was tested for the presence of Vpr by immunoprecipitation with Vpr-specific antibodies. The results of these experiments show that Vpr is trans-incorporated into virions but at a lower efficiency than when Vpr is expressed from a proviral construct. The minimal viral genetic information necessary for Vpr incorporation was a deleted provirus encoding only the pr55gag polyprotein precursor. Incorporation of Vpr requires the expression but not the processing of gag products and is independent of pol and env expression. Direct interaction of Vpr with the Pr55gag precursor protein was demonstrated by coprecipitation experiments with gag product-specific antibodies. Overall, these results indicate that HIV-1 Vpr is incorporated into the nascent virion through an interaction with the Gag precursor polyprotein and demonstrate a novel mechanism by which viral protein can be incorporated into virus particles.
Induction of beta interferon by human immunodeficiency virus type 1 and its gp120 protein in human monocytes-macrophages: role of beta interferon in restriction of virus replication
Authors: Gessani S , Puddu P , Varano B , Borghi P , Conti L , Fantuzzi L , Belardelli F
Published: January 2020

In vitro cultivated human monocytes show a time-dependent differentiation into macrophages, characterized by an increased expression of macrophage-specific antigens. Monocytes-macrophages were infected with human immunodeficiency virus type 1 strain Ba-L (HIV-1Ba-L) at different stages of differentiation. When 7-day cultured macrophages were infected in the presence of antibodies to beta interferon (IFN-beta), a significant increase in HIV-1 p24 release was detected. This effect was not observed in 1-day monocytes. This finding suggests that IFN-beta secreted by the infected macrophages inhibits p24 release. Treatment of cultured macrophages with recombinant gp120 (rgp120) protein resulted in the induction of IFN-beta mRNA and in an antiviral state to vesicular stomatitis virus. This rgp120-induced antiviral state was largely neutralized by antibodies to IFN-beta, whereas anti-IFN-alpha antibodies were ineffective. In cultured macrophages, 0.1 IU of IFN-beta per ml was sufficient to induce a marked inhibition of vesicular stomatitis virus yield, whereas this dose was ineffective in 1-day monocytes. These results indicate that (i) HIV-1 (possibly in part through its gp120 protein) induces low levels of IFN-beta in macrophages and (ii) this IFN-beta is very effective in inducing an antiviral state in differentiated macrophages.
A human cytomegalovirus early promoter with upstream negative and positive cis-acting elements: IE2 negates the effect of the negative element, and NF-Y binds to the positive element
Authors: Huang L , Malone C L , Stinski M F
Published: January 2020

The human cytomegalovirus early promoter for the UL4 gene, which codes for an early viral envelope glycoprotein designated gpUL4, requires immediate-early viral protein two (IE2) synthesis to be activated (C.-P. Chang, C. L. Malone, and M. F. Stinski, J. Virol. 63:281, 1989). We investigated the cis-acting and trans-acting factors that regulate transcription from this UL4 promoter. In transient transfection assays, the viral IE2 protein negated the effect of an upstream cis-acting negative element and enhanced downstream gene expression. A cis-acting positive element contributed to the activity of the viral promoter when an upstream cis-acting negative element was deleted or when the viral IE2 protein was present. The cellular protein(s) that binds to the cis-acting negative element requires further investigation. The cellular protein that binds to the cis-acting positive element was characterized. Two DNA sequence-specific protein complexes were detected with DNA probes spanning the region containing the cis-acting positive element and human cytomegalovirus-infected human fibroblast cell nuclear extracts. The more slowly migrating complex was labeled complex A, and the faster was labeled complex B. Only complex B was detected with mock-infected cell nuclear extracts. Competition experiments confirmed the specificity of the A and B complexes. The protein bound to the DNA in both the complexes contacts a CCAAT box imperfect dyad symmetry (5'CCAATCACTGG3'). Either CCAAT box within the dyad symmetry could compete for binding the nuclear factor. Mutation of the CCAAT box dyad symmetry resulted in a decrease of the transcriptional activity from the UL4 promoter. A cellular transcription factor, antigenically related to nuclear factor-Y (NF-Y), was found in both complexes A and B. Events associated with viral infection caused phosphorylation of protein complex A. Dephosphorylation of the DNA-binding protein converts complex A to complex B. The effect of phosphorylation of NF-Y is not known.
Suppression of rat cytomegalovirus replication by antibodies against gamma interferon
Authors: Haagmans B L , van der Meide P H , Stals F S , van den Eertwegh A J , Claassen E , Bruggeman C A , Horzinek M C , Schijns V E
Published: January 2020

The role of gamma interferon (IFN-gamma) in the resolution of rat cytomegalovirus (RCMV) infection was investigated. In the spleen, IFN-gamma-producing cells reached maximum numbers on day 7 after infection. Prophylactic treatment with high doses of recombinant rat IFN-gamma exerted antiviral activity in fibroblasts and protected immunosuppressed rats against a lethal RCMV challenge. Remarkably, in immunocompetent rats, neutralization of endogenous IFN-gamma activity significantly reduced the numbers of RCMV antigen-expressing cells in the spleen, the predominant site of viral replication. Moreover, protection of radiation-immunosuppressed infected rats by transferred immune T cells was enhanced by coinjection of IFN-gamma neutralizing antibodies. The observations were paralleled by in vitro findings: low concentrations of IFN-gamma enhanced viral replication in both macrophages and fibroblasts. These data suggest that IFN-gamma can play different and even opposite roles in the regulation of RCMV replication in vivo; T lymphocytes may contribute to the progression of RCMV infection by secreting IFN-gamma.
The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A)
Authors: Qiu Y , Krug R M
Published: January 2020

The influenza virus NS1 protein inhibits the nuclear export of a spliced viral mRNA, NS2 mRNA (F. V. Alonso-Caplen, M. E. Nemeroff, Y. Qiu, and R. M. Krug, Genes Dev. 6:255-267, 1992). To identify the sequence in NS2 mRNA that is recognized by the NS1 protein, we developed a gel shift assay for the formation of specific RNA-protein complexes. With this assay, it was established that the NS1 protein binds to the poly(A) sequence at the 3' end of NS2 mRNA and of other mRNAs. In addition, the NS1 protein was shown to bind to poly(A) itself. This specificity was also observed in vivo. The NS1 protein inhibited the nuclear export of every poly(A)-containing mRNA that was tested. In contrast, the NS1 protein failed to inhibit the nuclear export of an mRNA whose 3' end was generated by cleavage without subsequent addition of poly(A). Addition of poly(A) to this mRNA enabled the NS1 protein to inhibit mRNA export. The implications of these results for the role of the NS1 protein during virus infection are discussed.
Duck hepatitis B virus infection of Muscovy duck hepatocytes and nature of virus resistance in vivo
Authors: Pugh J C , Simmons H
Published: January 2020

To test the hypothesis that in vivo resistance to hepadnavirus infection was due to resistance of host hepatocytes, we isolated hepatocytes from Muscovy ducklings and chickens, birds that have been shown to be resistant to duck hepatitis B virus (DHBV) infection, and attempted to infect them in vitro with virus from congenitally infected Pekin ducks. Chicken hepatocytes were resistant to infection, but we were able to infect approximately 1% of Muscovy duck hepatocytes in culture. Infection requires prolonged incubation with virus at 37 degrees C. Virus spread occurs in the Muscovy cultures, resulting in 5 to 10% DHBV-infected hepatocytes by 3 weeks after infection. The relatively low rate of accumulation of DHBV DNA in infected Muscovy hepatocyte cultures is most likely due to inefficient spread of virus infection; in the absence of virus spread, the rates of DHBV replication in Pekin and Muscovy hepatocyte cultures are similar. 5-Azacytidine treatment can induce susceptibility to DHBV infection in resistant primary Pekin hepatocytes but appears to have no similar effect in Muscovy cultures. The relatively inefficient infection of Muscovy duck hepatocytes that we have described may account for the absence of a detectable viremia in Muscovy ducklings experimentally infected with DHBV.
Evidence for a functional interaction between the V1/V2 and C4 domains of human immunodeficiency virus type 1 envelope glycoprotein gp120
Authors: Freed E O , Martin M A
Published: January 2020

The domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein that are required for envelope function have been partially characterized. Little is known, however, about the nature of the interactions between these domains. To identify regions of the HIV-1 envelope glycoprotein that are involved in interactions necessary for proper envelope function, we constructed a series of 14 envelope recombinants between the env genes of two HIV-1 isolates. The envelope chimeras were examined for their ability to induce syncytia, to be proteolytically processed, and to function during a spreading viral infection. Our results demonstrate that the exchange between the two isolates of the first and second hypervariable regions (V1/V2) of gp120 results in defects in envelope glycoprotein processing, syncytium formation, and infectivity. Long-term passage of cultures infected with virus bearing a V1/V2 chimeric envelope glycoprotein leads to the emergence of a revertant virus with replication characteristics comparable to those of the wild type. Analysis of the revertant indicated that an Ile-->Met change in the C4 region of gp120 (between hypervariable regions V4 and V5) is responsible for the revertant phenotype. This single amino acid change restores infectivity without significantly affecting gp160 processing, CD4 binding, or the levels of virion-associated gp120. While the Ile-->Met change in C4 greatly enhances the fusogenic potential of the V1/V2 chimeric envelope glycoprotein, it has a detrimental effect on syncytium formation when analyzed in the context of the wild-type envelope. These results suggest that an interaction required for proper envelope glycoprotein function occurs between the V1/V2 and C4 regions of gp120.
Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids
Authors: Zhou W , Parent L J , Wills J W , Resh M D
Published: January 2020

Retroviral Gag proteins are targeted to the plasma membrane, where they play the central role in virion formation. Several studies have suggested that the membrane-binding signal is contained within the amino-terminal matrix sequence; however, the precise location has never been determined for the Gag protein of any retrovirus. In this report, we show that the first 31 residues of human immunodeficiency virus type 1 Gag protein can function independently as a membrane-targeting domain when fused to heterologous proteins. A bipartite membrane-targeting motif was identified, consisting of the myristylated N-terminal 14 amino acids and a highly basic region that binds acidic phospholipids. Replacement of the N-terminal membrane-targeting domain of pp60v-src with that of human immunodeficiency virus type 1 Gag elicits efficient membrane binding and a transforming phenotype. Removal of myristate or the basic region results in decreased membrane binding of Gag-Src chimeras in vitro and impaired virion formation by Pr55gag in vivo. We propose that the N-terminal Gag sequence functions as a targeting signal to direct interaction with acidic phospholipids on the cytoplasmic leaflet of the plasma membrane.
Variability of the env gene in cynomolgus macaques persistently infected with human immunodeficiency virus type 2 strain ben
Authors: Tolle T , Petry H , Bachmann B , Hunsmann G , Lüke W
Published: January 2020

The sequence variability of distinct regions of the proviral env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben) isolated sequentially over 3 to 4 years from six experimentally infected macaques was studied. The regions investigated were homologous to the V1, V2, V3, V4, V5, and V7 hypervariable regions identified in the env genes of HIV-1 and simian immunodeficiency virus SIVmac, respectively. In contrast to findings with HIV-1 and SIVmac, the V1- and V2-homologous regions were found to be highly conserved during the course of the HIV-2ben infection in macaques. The V3-homologous region showed a degree of variation comparable to that of HIV-1 but not of SIV. In the V4-, V5-, and V7-homologous regions, mutation hot spots were detected in most reisolates of the infected monkeys. Most of these mutations occurred during the first 10 weeks after infection. After 50 weeks, new mutations were rarely detected. At most mutation sites, a dynamic equilibrium between the mutated viral isotype and the infecting predominant wild type was present. This equilibrium might prevent an accumulation of mutations in isolates later in the course of infection.
In vivo replicative status and envelope heterogeneity of equine infectious anemia virus in an inapparent carrier
Authors: Kim C H , Casey J W
Published: January 2020

The distribution and replicative status of equine infectious anemia virus (EIAV) DNA in the tissues of a well-characterized inapparent carrier horse were established by using the PCR technique. The EIAV pol region could be amplified in all of the tissues tested, including the cerebellum and periventricular tissue, at concentrations approximately 10(5)-fold less than in the same tissue from an acutely infected horse. Further analysis of the EIAV genome, with primer pairs diagnostic for sequential stages of reverse transcription, suggests that EIAV DNA in the brain, liver, and lymph nodes was incompletely synthesized. The products of reverse transcription were found to diminish progressively during first-strand synthesis, while products indicative of second-strand synthesis were observed only in kidney and spleen DNA samples. Sequences specific for different regions of the envelope could not be amplified from any of the tissues of the inapparent carrier, suggesting that the envelope is highly variable and may be subject to extensive drift. Together, the data suggest that low levels of EIAV replication persist without causing clinical disease in an inapparent carrier.
Structure and transcription of an immediate-early region in the human herpesvirus 6 genome
Authors: Schiewe U , Neipel F , Schreiner D , Fleckenstein B
Published: January 2020

The unique segment of the human herpesvirus 6 (HHV-6) genome is essentially collinear to the unique long DNA segment of another betaherpesvirus, the human cytomegalovirus (HCMV). However, the HHV-6 genomic section that is analogous in position to the major immediate-early (IE) locus of HCMV does not exhibit recognizable sequence homologies. The respective HHV-6 region of 5.5 kbp is flanked on one side by 25 to 28 incomplete tandem repeats of 105 to 110 bp that contain, with one exception, a single KpnI restriction site (KpnI repeats). About 250 reiterations of the sequence motif CACATA are located on the other end. We identified two open reading frames of 375 and 2,595 nucleotides, respectively, on one strand. Strand-specific Northern blot analyses with RNA harvested from HHV-6 (strain U1102)-infected HSB-2 cells or cord blood lymphocytes revealed two transcripts of about 3.5 and 4.7 kb in the corresponding orientation. Sequence analyses of the respective cDNA clones and primer extension experiments were used to map the mRNAs. The two transcripts are coterminal and multiply spliced and code for the same putative 104.6-kDa protein, but they are initiated from different promoters. Characterization of smaller cDNA clones and Northern blotting with other strand-specific probes showed that singly spliced mRNAs of 1.0 and 1.5 kb are transcribed from the opposite strand; they could code for a 17.2-kDa polypeptide. Blocking experiments with cycloheximide led to the conclusion that only the 3.5-kb mRNA is synthesized in the absence of protein biosynthesis upon infection with cell-free virus. This identifies a single IE gene of HHV-6 at the genomic position corresponding to the major IE region of HCMV, although the coding content and transcriptional regulation are quite different for these two herpesvirus IE regions.
A domain of murine retrovirus surface protein gp70 mediates cell fusion, as shown in a novel SC-1 cell fusion system
Authors: Andersen K B
Published: January 2020

Virus-induced cell fusion of the fusion-from-without type was observed in SC-1 cells infected with Moloney murine leukemia virus when grown in NIH 3T3 cells. Replication-competent virus mutants with altered surface protein gp70 were examined. Fusion mutations were found in the proline-rich region of gp70. They acted on a step after binding and before or during endocytosis. The fusion mutants had an altered gp70 isomer pattern, presumably caused by different glycosylation. Other mutants with deleted glycans were analyzed, and some which also showed defective fusion were found. The interrelationship of the proline-rich region, glycosylation, and fusion is discussed.
Identification of a dimerization domain in the C-terminal segment of the IE110 transactivator protein from herpes simplex virus
Authors: Ciufo D M , Mullen M A , Hayward G S
Published: January 2020

The 775-amino-acid IE110 (or ICP0) phosphoprotein of herpes simplex virus (HSV) functions as an accessory transcription factor during the lytic cycle and plays a critical role in reactivation from latent infection. By immunofluorescence analysis, IE110 localizes in a novel pattern consisting of several dozen spherical punctate granules in the nuclei of DNA-transfected cells. We constructed a hybrid version of IE110 that contained an epitope-tagged domain from the N terminus of the HSV IE175 protein and lacked the IE110 N-terminal domain that confers punctate characteristics. This hybrid IE175(N)/IE110(C) protein gave an irregular nuclear diffuse pattern on its own but was redistributed very efficiently into spherical punctate granules after cotransfection with the wild-type HSV-1 IE110 protein. Similar colocalization interactions occurred with internally deleted forms of IE110 that lacked the zinc finger region or large segments from the center of the protein, including both cytoplasmic and elongated punctate forms, but C-terminal truncated versions of IE110 did not interact. In all such interactions, the punctate phenotype was dominant. Evidence that C-terminal segments of IE110 could also form stable mixed-subunit oligomers in vitro was obtained by coimmunoprecipitation of in vitro-translated IE110 polypeptides with different-size hemagglutinin epitope-tagged forms of the protein. This occurred only when the two forms were cotranslated, not when they were simply mixed together. An in vitro-synthesized IE110 C-terminal polypeptide also gave immunoprecipitable homodimers and heterodimers when two different-size forms were cross-linked with glutaraldehyde and reacted specifically with a bacterial glutathione S-transferase/IE110 C-terminal protein in far-Western blotting experiments. The use of various N-terminal and C-terminal truncated forms of IE110 in the in vivo assays revealed that the outer boundaries of the interaction domain mapped between codons 617 and 711, although inclusion of adjacent codons on either side increased the efficiency severalfold in some assays. We conclude that the C-terminal region of IE110 contains a high-affinity self-interaction domain that leads to stable dimer and higher-order complex formation both in DNA-transfected cells and in in vitro assays. This segment of IE110 is highly conserved between HSV-1 and HSV-2 and appears to have the potential to play an important role in the interaction with the IE175 protein, as well as in correct intracellular localization, but it is not present in the equivalent proteins from varicella-zoster virus, pseudorabies virus, or equine abortion virus.
Analysis of antibody responses to predominant linear epitopes of Theiler's murine encephalomyelitis virus
Authors: Inoue A , Choe Y K , Kim B S
Published: January 2020

Using synthetic peptides, we have defined the major linear antibody epitopes of Theiler's murine encephalomyelitis virus (TMEV), i.e., A1A (VP1(12-25)), A1Ba (VP1(146-160)), A1Cb (VP1(262-276)), A2A (VP2(2-16)), A2B (VP2(165-179)), and A3A (VP3(24-37)). A time course study with either pooled or individual sera indicates that susceptible SJL mice intracerebrally infected with TMEV strongly and selectively recognize the A1Cb epitope of VP1, compared with resistant BALB/c or C57BL/6 mice, which broadly recognize most of the epitopes on the different capsid proteins. However, antibodies from SJL mice subcutaneously immunized with TMEV recognize primarily A1Ba, A1Cb, and A2A epitopes. A similar predominant recognition of the A1Cb epitope was found with antibodies from the cerebrospinal fluid of intracerebrally virus-infected SJL mice. Interestingly, a substantial level of antibodies against the A1Cb epitope in virus-infected SJL mice is of the immunoglobulin G2a subclass, in contrast to an undetectable level of this immunoglobulin G subclass in virus-immunized SJL mice. The level of in vitro viral neutralization by antibodies did not correlate with the clinical signs. Antibodies to A1Cb, A2A, and A2B were able to neutralize viral plaque formation in vitro, while antibodies to A3A, A1A, and A1Ba were not.
Mapping and characterization of antigenic epitopes and the nucleic acid-binding domains of the VP6 protein of bluetongue viruses
Authors: Hayama E , Li J K
Published: January 2020

Heterologously expressed VP6 and truncated VP6 proteins of bluetongue virus (BTV) serotype 11 purified to near homogeneity were used for structure and function analyses. The yield of the expressed VP6 was host cell dependent. Six antigenic epitopes of VP6 of BTV were identified and mapped by immunoblot analyses and enzyme-linked immunosorbent assay with oligoclonal antibodies. These determinants were surface accessible and conserved among the cognate VP6 proteins of five U.S. BTV serotypes. The amino acid sequences and sizes of these six antigenic epitopes were determined, and their precise locations were also mapped and confirmed by deletion analyses. The nucleic acid binding activities of VP6, confirmed by electrophoretic mobility shift assay, were concentration dependent. The binding activities and affinities of the purified expressed VP6 protein towards double-stranded RNA and double-stranded DNA were similar. Two domains of VP6, corresponding to three of the six antigenic epitopes, were responsible for the nucleic acid binding activities and have been mapped within 28 amino acids near the middle and 11 residues near the carboxyl terminus of VP6 by electrophoretic mobility shift assay and deletion mutant analyses. Synthetic oligopeptides corresponding to these three regions also exhibited similar concentration-dependent nucleic acid binding activities.
Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants
Authors: Jones T R , Sun L , Bebernitz G A , Muzithras V P , Kim H J , Johnston S H , Baum E Z
Published: January 2020

The human cytomegalovirus UL80 open reading frame encodes protease and assembly protein from its N- and C-terminal regions, respectively. We reported previously that a 30-kDa protease is derived by autoproteolytic processing of a polyprotein which is the translation product of the entire UL80 open reading frame (E. Z. Baum, G. A. Bebernitz, J. D. Hulmes, V. P. Muzithras, T. R. Jones, and Y. Gluzman, J. Virol. 67:497-506, 1993). Three autoproteolytic cleavage sites within the UL80 polyprotein were characterized; site 143 is within the protease domain and inactivates the protease. In this article, we report (i) expression analyses of UL80 in infected cells, including the processing kinetics of the UL80 polyprotein; (ii) the existence of an additional cleavage site (site 209) within the protease domain of the UL80 polyprotein; and (iii) the effect of mutagenesis at each of the cleavage sites upon proteolytic activity and steady-state levels of the UL80 processing products. During the course of infection, UL80 polyprotein processing begins at cleavage site 643 and follows at sites 256 and 143. Cleavage at site 643 and/or 256 within the polyprotein is not a prerequisite for efficient protease activity, since all three proteases (85-, 80-, and 30-kDa proteins) were equally active in cleaving the assembly protein precursor to its mature form. Inhibition of cleavage at site 143 resulted in a three- to sixfold increase in the steady-state level of the 30-kDa protease, supporting the hypothesis that cleavage at this site may represent a mechanism by which cytomegalovirus regulates the level of active protease.
Comparison of 5' and 3' long terminal repeat promoter function in human immunodeficiency virus
Authors: Klaver B , Berkhout B
Published: January 2020

The architecture of a retroviral genome presents some unusual features for transcriptional regulation because of duplication of the transcriptional control sequences in the 5' and 3' long terminal repeats (LTRs). We have studied the transcriptional activity of the 5' and 3' LTRs of human immunodeficiency virus type 1 (HIV-1) vectors. Using full-length HIV molecular clones, we demonstrate that both LTRs function as Tat-inducible promoters. However, the absolute levels of transcription were found to be much higher for the 5' LTR than for the 3' LTR promoter. When transcription was assayed for an integrated HIV-1 provirus, we also found that the upstream 5' LTR element was the major transcriptional promoter. 3' LTR transcription, however, can be triggered by inactivation of the 5' LTR promoter. Likewise, 5' LTR transcription is induced in constructs lacking a functional 3' LTR promoter. This phenomenon of promoter suppression may have important implications for the design of HIV-based retrovirus vectors.
Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes
Authors: Parolin C , Dorfman T , Palú G , Göttlinger H , Sodroski J
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) can be used to generate recombinant viral vectors for delivery of heterologous genes to human CD4-positive lymphocytes. To define the cis-acting sequences required for efficient gene transfer, a number of HIV-1 vectors containing a previously identified packaging signal, long terminal repeats, and additional gag, pol, and env viral sequences were designed. By providing the viral proteins in trans, recombinant viruses were generated and analyzed for their abilities to transfer genes into human T lymphocytes. Inclusion of up to 653 nucleotides derived from the 5' end of the gag gene in the vector improved the efficiency of gene transfer, but inclusion of additional gag or pol sequences did not further improve this efficiency. The increased efficiency of gene transfer associated with the inclusion of 5' gag sequences in the vector arose, at least in part, from an increase in the packaging of vector RNA. The presence of the Rev-responsive element (RRE) increased the efficiency of transfer of vectors containing significant lengths of gag sequence, as expected from the Rev requirement for nucleus-to-cytoplasm transport of unspliced vector RNA containing intact packaging signals. However, the presence of a RRE did not affect the transfer efficiency of smaller vectors lacking significant lengths of gag sequences, arguing against a specific role for the RRE in packaging or vector transfer. These results contribute to an understanding of the minimal cis-acting sequences that operate in the context of HIV-1 vectors for delivering genes into human lymphocytes.
Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates
Authors: Chow S A , Brown P O
Published: January 2020

The integrase encoded by human immunodeficiency virus type 1 (HIV-1) is required for integration of viral DNA into the host cell chromosome. In vitro, integrase mediates a concerted cleavage-ligation reaction (strand transfer) that results in covalent attachment of viral DNA to target DNA. With a substrate that mimics the strand transfer product, integrase carries out disintegration, the reverse of the strand transfer reaction, resolving this integration intermediate into its viral and target DNA parts. We used a set of disintegration substrates to study the catalytic mechanism of HIV-1 integrase and the interaction between the protein and the viral and target DNA sequence. One substrate termed dumbbell consists of a single oligonucleotide that can fold to form a structure that mimics the integration intermediate. Kinetic analysis using the dumbbell substrate showed that integrase turned over, establishing that HIV-1 integrase is an enzyme. Analysis of the disintegration activity on the dumbbell substrate and its derivatives showed that both the viral and target DNA parts of the molecule were required for integrase recognition. Integrase recognized target DNA asymmetrically: the target DNA upstream of the viral DNA joining site played a much more important role than the downstream target DNA in protein-DNA interaction. The site of transesterification was determined by both the DNA sequence of the viral DNA end and the structure of the branched substrate. Using a series of disintegration substrates with various base modifications, we found that integrase had relaxed structural specificity for the hydroxyl group used in transesterification and could tolerate distortion of the double-helical structure of these DNA substrates.