bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcellular distribution of human immunodeficiency virus type 1 Rev and colocalization of Rev with RNA splicing factors in a speckled pattern in the nucleoplasm
Authors: Kalland K H , Szilvay A M , Langhoff E , Haukenes G
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) Rev (regulator of virion protein expression) protein exemplifies a new type of posttranscriptional regulation. One main function of Rev is to increase the cytoplasmic expression of unspliced and incompletely spliced retroviral mRNAs from which viral structural proteins are made. In that way, Rev is essential in order to complete the retroviral life cycle. The biology of Rev in the host cell has remained elusive. In this study, a complex distribution of Rev in single cells was found. Rev was found in the cytoplasm, in a perinuclear zone, in the nucleoplasm, and in the nucleoli. In the nucleoplasm, Rev colocalized in a speckled pattern with host cell factors known to assemble on nascent transcripts. Those factors are involved in the processing of heterogeneous RNA to spliced mRNA in the nucleoplasm of all cells. The distribution of Rev was dependent only on Rev and host cell interactions, since neither the Rev target RNA nor other HIV proteins were expressed in the cells. Rev was found in the same subcellular compartments of cells treated for extended periods with cycloheximide, an inhibitor of protein synthesis. This finding implies that Rev shuttles continuously between cytoplasmic and nucleoplasmic compartments. The results suggest a potential role for Rev both in the RNA-splicing process and in the nucleocytoplasmic transport of Rev-dependent HIV mRNA.
Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro
Authors: Svitkin Y V , Meerovitch K , Lee H S , Dholakia J N , Kenan D J , Agol V I , Sonenberg N
Published: January 2020

Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of trans-acting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNA-programmed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.
Identification and characterization of a human herpesvirus 6 gene segment capable of transactivating the human immunodeficiency virus type 1 long terminal repeat in an Sp1 binding site-dependent manner
Authors: Wang J , Jones C , Norcross M , Bohnlein E , Razzaque A
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) is transactivated by various extracellular signals and viral cofactors that include human herpesviruses. These transactivators are capable of transactivating the HIV-1 LTR through the transactivation response element, NF-kappa B, or other regulatory binding elements. Human herpesvirus 6 (HHV-6) is a potential cofactor of HIV-1. Here, we report that an HHV-6 gene segment, ZVH14, which can neoplastically transform NIH 3T3 and human keratinocytes, is capable of transactivating HIV-1 LTR chloramphenicol acetyltransferase constructs in an Sp1 binding site-dependent manner. Transactivation increased synergistically in the presence of multiple Sp1 sites and was dramatically reduced by cotransfection with oligomers designed to form triplex structures with HIV-1 LTR Sp1 binding sites. HIV-1 LTR NF-kappa B sites were not essential for ZVH14-mediated transactivation. A putative open reading frame in ZVH14, B115, which may encode a highly basic peptide consisting of 115 amino acid residues, showed transactivation capacity similar to that of ZVH14. This open reading frame also transactivated the HIV-1 LTR in an Sp1 site-dependent fashion in African green monkey kidney cells and human T cells. These data suggest that HHV-6 may stimulate HIV-1 replication via transactivation of Sp1 binding sites present in the HIV-1 promoter.
Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein
Authors: Rein A , Mirro J , Haynes J G , Ernst S M , Nagashima K
Published: January 2020

In the murine leukemia viruses (MuLVs), the Env complex is initially cleaved by a cellular protease into gp70SU and pre15ETM. After the virus particle is released from the cell, the C-terminal 16 residues are removed from the cytoplasmic domain of pre15E by the viral protease, yielding the mature p15ETM and p2E. We have investigated the function of this cleavage by generating a Moloney MuLV mutant, termed p2E-, in which the Env coding region terminates at the cleavage site. This mutant synthesizes only the truncated, mature form of TM rather than its extended precursor. When cells expressing this truncated Env protein are cocultivated with NIH 3T3 cells, they induce rapid cell-cell fusion. Thus, the truncated form, which is normally found in virions but not in virus-producing cells, is capable of causing membrane fusion. We conclude that the 16-residue p2E tail inhibits this activity of Env until the virus has left the cell. p2E- virions were found to be infectious, though with a lower specific infectivity than that of the wild type, showing that p2E does not play an essential role in the process of infection. Fusion was also observed with a chimeric p2E- virus in which gp70SU and nearly all of p15ETM are derived from amphotropic, rather than Moloney, MuLV. In a second mutant, an amino acid at the cleavage site was changed. The pre15E protein in this mutant is not cleaved. While the mutant Env complex is incorporated into virions, these particles have a very low specific infectivity. This result suggests that the cleavage event is essential for infectivity, in agreement with the idea that removal of p2E activates the membrane fusion capability of the Env complex.
Induction of HLA class I and class II expression in human T-lymphotropic virus type I-infected neuroblastoma cells
Authors: Lehky T J , Cowan E P , Lampson L A , Jacobson S
Published: January 2020

Human T-lymphotropic virus type I (HTLV-I) is associated with a neurologic disease, HTLV-I-associated myelopathy-tropical spastic paraparesis, in which both pathological and immunological changes are observed within the central nervous system. The pathogenesis of infection in HTLV-I-associated myopathy-tropical spastic paraparesis is not well understood with respect to the cell tropism of HTLV-I and its relationship to the destruction of neural elements. In this study, neuroblastoma cells were infected with HTLV-I by coculturing with HUT-102 cells to demonstrate that cells of neuronal origin are susceptible to this retroviral infection. HTLV-I infection of the neuroblastoma cells was confirmed by verifying the presence of HTLV-I gp46 surface antigens by flow cytometry and by verifying the presence of HTLV-I pX RNA by Northern (RNA) blotting and in situ hybridization techniques. To determine whether HTLV-I infection could potentially lead to changes in cell surface recognition by the immune system, the infected neuroblastoma cells were analyzed for altered HLA expression. The HTLV-I-infected, cocultured neuroblastoma cells were shown, through cell surface antigen expression and RNA transcripts, to express HLA classes I and II. In contrast, cocultured neuroblastoma cells that did not become infected with HTLV-I expressed only HLA class I. HLA class I expression was enhanced by the cytokines tumor necrosis factor alpha and gamma interferon and in the presence of HUT-102 supernatant. In this system, expression of HLA class I and II molecules appeared to be regulated by different mechanisms. HLA class I expression was probably induced by cytokines present in the HUT-102 supernatant and was not dependent on HTLV-I infection. HLA class II expression required HTLV-I infection of the cells. The observation of HTLV-I infection leading to HLA induction in these neuroblastoma cells provides a possible mechanism for immunologic recognition of infected neuronal cells.
Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis
Authors: Shirako Y , Strauss J H
Published: January 2020

Nonstructural proteins of Sindbis virus, nsP1, nsP2, nsP3, and nsP4, as well as intermediate polyproteins, are produced from two precursor polyproteins, P123 and P1234, by a proteolytic enzyme encoded in the C-terminal half of nsP2. We studied the requirements for and the functions of the intermediate and mature processing products for Sindbis virus RNA synthesis by using site-directed mutants which have a defect(s) in processing the 1/2, 2/3, or 3/4 cleavage sites either singly or in various combinations. A mutant defective in cleaving both the 1/2 and 2/3 sites, which makes only uncleavable P123 and mature nsP4 as final products, produced 10(-3) as much virus as did the wild-type virus after 10 h at 30 degrees C and was nonviable at 40 degrees C. A mutant defective in processing the 2/3 site, which makes nsP1, nsP4, and P23 as well as precursor P123, grew 10(-1) as efficiently as wild-type virus at 30 degrees C and 10(-3) as efficiently at 40 degrees C. Early minus-strand RNA synthesis by these mutants was as efficient as that by wild-type virus, whereas plus-strand RNA synthesis was substantially decreased compared with that by wild-type virus. A mutant defective in processing the 3/4 site was nonviable at either 30 or 40 degrees C. The 3/4 site mutant could be complemented by the mutant unable to cleave either the 1/2 or 2/3 site, which can provide mature nsP4. We interpret these results to signify that (i) mature nsP4 is required for RNA replication, (ii) nsP4 and uncleaved P123 function in minus-strand RNA synthesis, and (iii) cleavage of P123 is required for efficient plus-strand RNA synthesis. We propose that Sindbis virus RNA replication is regulated by differential proteolysis of P123. Early in infection, nsP4 and uncleaved P123 form transient minus-strand RNA replication complexes which vanish upon cleavage of P123. Later in infection, an elevated level of viral proteinase activity eliminates de novo synthesis of P123, and no further synthesis of minus-strand RNA is possible. In contrast, nsP4 and cleavage products from P123 form plus-strand RNA replication complexes which are stable and remain active throughout the infection cycle.
Murine AIDS is initiated in the lymph nodes draining the site of inoculation, and the infected B cells influence T cells located at distance, in noninfected organs
Authors: Simard C , Huang M , Jolicoeur P
Published: January 2020

The infection of cells which belong to the B-cell lineage is thought to be the primary event leading to the phenotypic and functional alterations seen in the murine AIDS (M. Huang, C. Simard, D. Kay, and P. Jolicoeur, J. Virol. 65:6562-6571, 1991). Using in situ hybridization, we studied the time course of the anatomic distribution of the murine AIDS-infected B cells in C57BL/6 mice inoculated intraperitoneally or in the foot pad with helper-free stocks of the defective murine AIDS virus. The local lymph nodes draining the injection site (the mediastinal or popliteal lymph nodes) were the primary organs in which infected B cells could be detected. From this initial site, the proliferating infected B cells were found to migrate progressively to most of the other lymph nodes and to the spleen. The bone marrow cells (containing the precursor B cells) were not found to be infected by the virus. These results suggest that the defective murine AIDS virus infects mature Ly-1- B cells present in lymph nodes. We compared the concanavalin A response of the T cells at an early time postinoculation, before all lymphoid organs are infiltrated with infected B cells. In lymphoid organs free of infected B cells, T cells were found to be hyperresponsive. In lymphoid organs in which infected B cells were present, T cells were hyporesponsive. These data suggest that infected B cells influence distant T cells, maybe by the release of a circulating factor or through another uninfected cell population activated by the infected B cells.
The activity of the pseudorabies virus latency-associated transcript promoter is dependent on its genomic location in herpes simplex virus recombinants as well as on the type of cell infected
Authors: Huang C J , Rice M K , Devi-Rao G B , Wagner E K
Published: January 2020

As do many other alphaherpesviruses, pseudorabies virus (PRV) transcribes a limited portion of its viral genome in latently infected neurons during latency. The sequence of the PRV latency-associated transcript (LAT) is bounded on its 5' end by a putative promoter region which contains sequence elements similar to those characterized for the herpes simplex virus (HSV) LAT promoter. Using the bacterial beta-galactosidase gene as a reporter, we have assayed PRV LAT promoter activity in the genomic environment in recombinant HSVs. The PRV LAT promoter-beta-galactosidase reporter gene was recombined into the terminal and internal long repeat regions (RL regions), replacing the normal HSV LAT promoter, the cap site, and the first 60 bases of the primary transcript. When recombined into the RL region, appreciable reporter gene expression was observed following infection of two cell lines of neuronal origin; little or no activity was seen with these recombinants following infection of rabbit skin or mouse embryo fibroblasts. No significant expression was seen when the promoter was recombined into the gC locus in the long unique region in any of the cell types utilized. Such results suggest that the PRV latency promoter contains neuronal cell-specific elements and that the HSV RL region provides an appropriate genomic environment for the manifestation of that specificity.
Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2-symmetric protease inhibitor
Authors: Ho D D , Toyoshima T , Mo H , Kempf D J , Norbeck D , Chen C M , Wideburg N E , Burt S K , Erickson J W , Singh M K
Published: January 2020

Inhibitors of the human immunodeficiency virus type 1 protease represent a promising class of antiviral drugs for the treatment of AIDS, and several are now in clinical trials. Here, we report the in vitro selection of viral variants with decreased sensitivity to a C2-symmetric protease inhibitor (A-77003). We show that a single amino acid substitution (Arg to Gln or Lys) at position 8 of the protease results in a substantial decrease in the inhibitory activity of the drug on the enzyme and a comparable increase in viral resistance. These findings, when analyzed by using the three-dimensional structure of the protease-drug complex, provide a strategic guide for the future development of inhibitors of the human immunodeficiency virus type 1 protease.
Upstream U3 sequences in simian immunodeficiency virus are selectively deleted in vivo in the absence of an intact nef gene
Authors: Kirchhoff F , Kestler H W , Desrosiers R C
Published: January 2020

Major transcriptional control elements are located within the U3 region of the long terminal repeats (LTRs) of lentivirus and other retroviral genomes. The nef auxiliary gene of simian immunodeficiency virus (SIV) and human immunodeficiency virus overlaps about 70% of the 450- to 560-bp-long U3 region present in these primate lentiviruses. We analyzed viral DNA sequences present in rhesus monkeys infected with a mutant of SIVmac containing a 182-bp deletion in the region of nef that does not overlap the LTR. Between 50 and 100% of the viral DNA molecules in eight of nine monkeys infected for 16 or more months contained additional deletions of 111 to 302 bp within the 517-bp U3 region. These deletions were contained within a 334-bp region of U3 that is overlapped by the nef reading frame, and they did not affect the polypurine tract, the NF-kappa B binding site, or other sequence elements in this same region that are important for transcription and replication. Such deletions were not detected in any of 41 PCR amplifications from 8 rhesus monkeys infected with wild-type SIV for 8 to 26 months, nor were they detected in 10 animals infected with vpr, vpx or vpr-vpx deletion mutants. These results indicate that, in the absence of an intact nef gene, these upstream U3 sequences are not advantageous for the virus.
Two regions of an avian hepadnavirus RNA pregenome are required in cis for encapsidation
Authors: Calvert J , Summers J
Published: January 2020

We have constructed a series of deletion mutants spanning the genome of duck hepatitis B virus in order to determine which regions of the viral genome are required in cis for packaging of the pregenome into capsid particles. Deletion of sequences within either of two nonadjacent regions prevented replication of the mutant viral genomes expressed in a permissive avian hepatoma cell line in the presence of functionally active viral core and P proteins. Extraction of RNA from cells transfected with these replication-defective mutants showed that the mutants retained the capacity to be transcribed into a pregenomic-size viral RNA, but that these RNA species were not packaged into viral capsids. The two regions defined by these deletions are located 36 to 126 (region I) and 1046 to 1214 (region II) nucleotides downstream of the 5' end of the pregenome and contain sequences which are required in cis for encapsidation of the duck hepatitis B virus pregenome.
Mutation of either of two cysteine residues or deletion of the amino or carboxy terminus of nonstructural protein NS1 of bluetongue virus abrogates virus-specified tubule formation in insect cells
Authors: Monastyrskaya K , Booth T , Nel L , Roy P
Published: January 2020

Virus-specific tubules are characteristic of orbivirus infections and are likely to play an important role in virus morphogenesis. It has been shown that for bluetongue virus (BTV), the prototype orbivirus in the family Reoviridae, the virus-encoded NS1 protein forms tubules in insect cells when the BTV segment M6 gene is expressed by using a baculovirus vector. To understand the function of NS1 tubules and to identify the sequences involved in their polymerization, a series of mutant NS1 genes was generated and expressed in insect cell cultures by using baculovirus vectors. Three of the mutants were deletion mutants. One (AcNS1.dNT10) lacked 10 of the amino-terminal amino acids, and the other two mutants (AcNS1.dCT20 and AcNS1.dCT43) lacked 20 or 43 of the carboxy-terminal amino acids. In addition, site-directed mutants were constructed in which various single cysteines or pairs of cysteines were changed to serines. The ability of each mutant protein to form tubules was investigated. None of the deletion mutants formed tubules. The constructs in which the cysteines at amino acid positions 337 and/or 340 were replaced by serines (e.g., AcNS1.C337S,C340S) also did not form tubules. Instead, the NS1 protein of these and the deletion mutants made ribbon-like structures which formed large aggregates. Mutations involving six other cysteines (i.e., AcNS1.C37S,C43S,AcNS1.C462S,C465S, AcNS1.C104S, and AcNS1.C364S) produced tubules. The results show that both the amino and carboxy termini of the NS1 protein molecule and the cysteines at residues 337 and 340 are essential for tubule formation.
Activation of cerebral cytokine gene expression and its correlation with onset of reactive astrocyte and acute-phase response gene expression in scrapie
Authors: Campbell I L , Eddleston M , Kemper P , Oldstone M B , Hobbs M V
Published: January 2020

The pathogenesis of scrapie, a transmissible subacute spongiform encephalopathy, is unclear. However, certain aspects of the known cellular and molecular neuropathology in scrapie led us to hypothesize that cytokines could mediate cerebral pathological changes in this neurodegenerative disease. Therefore, expression of multiple cytokine genes in the brain and peripheral organs of scrapie-infected mice was examined. Late in the course of scrapie, expression of tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and IL-1 beta mRNA was markedly increased in the brain but not the spleen, kidneys, or liver. In time course studies, scrapie-infected mice exhibited increased cerebral expression of the TNF-alpha, IL-1 alpha, and IL-1 beta mRNAs by week 15 postinoculation--a time point that coincided with the onset of clinical symptoms. Thereafter, the levels of these cytokine transcripts increased progressively to the terminal stages of of the disease (week 25). To determine the relationship of the increased cerebral expression of the cytokine mRNAs to the development of pathological changes in scrapie, we examined the expression of the glial fibrillary acidic protein gene (a marker for astrocytosis) and the murine acute-phase response gene homologous to the alpha 1-antichymotrypsin gene (designated EB22/5.3). Markedly increased expression of both the glial fibrillary acidic protein and EB22/5.3 mRNAs was observed in the brain but not the peripheral organs of scrapie-infected mice. The increased expression of both these gene products also occurred at week 15 of infection and, thereafter, increased progressively to the terminal stages of the disease. Therefore, infection of mice with scrapie resulted in significant increases in the expression of the TNF-alpha, IL-1 alpha, and IL-1 beta gene products, whose pattern correlated with the onset and development of molecular and clinical pathological changes. Since scrapie is known not to evoke an immune response, the present findings strongly suggest the existence of a localized cerebral host response to the agent during which proinflammatory cytokines could be key pathogenic mediators.
An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs
Authors: Birnbaum M J , Clem R J , Miller L K
Published: January 2020

Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV (R. J. Clem, M. Fechheimer, and L. K. Miller, Science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV) (N. E. Crook, R. J. Clem, and L. K. Miller, J. Virol. 67:2168-2174, 1993). Using a genetic complementation assay to identify additional genes which inhibit apoptosis during infection with a p35 mutant, we have isolated a gene from Orgyia pseudotsugata NPV (OpMNPV) that was able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicted a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs were found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appeared to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA.
A transcriptional regulatory element is associated with a nuclease-hypersensitive site in the pol gene of human immunodeficiency virus type 1
Authors: Van Lint C , Ghysdael J , Paras P , Burny A , Verdin E
Published: January 2020

Analysis of the chromatin organization of the integrated human immunodeficiency virus type 1 (HIV-1) genome has previously revealed a major constitutive DNase I-hypersensitive site associated with the pol gene (E. Verdin, J. Virol. 65:6790-6799, 1991). In the present report, high-resolution mapping of this site with DNase I and micrococcal nuclease identified a nucleosome-free region centered around nucleotides (nt) 4490 to 4766. A 500-bp fragment encompassing this hypersensitive site (nt 4481 to 4982) exhibited transcription-enhancing activity (two- to threefold) when it was cloned in its natural position with respect to the HIV-1 promoter after transient transfection in U937 and CEM cells. Using in vitro footprinting and gel shift assays, we have identified four distinct binding sites for nuclear proteins within this positive regulatory element. Site B (nt 4519 to 4545) specifically bound four distinct nuclear protein complexes: a ubiquitous factor, a T-cell-specific factor, a B-cell-specific factor, and the monocyte/macrophage- and B-cell-specific transcription factor PU.1/Spi-1. In most HIV-1 isolates in which this PU box was not conserved, it was replaced by a binding site for the related factor Ets1. Factors binding to site C (nt 4681 to 4701) had a DNA-binding specificity similar to that of factors binding to site B, except for PU.1/Spi-1. A GC box containing a binding site for Sp1 was identified (nt 4623 to 4631). Site D (nt 4816 to 4851) specifically bound a ubiquitously expressed factor. These results identify a transcriptional regulatory element associated with a nuclease-hypersensitive site in the pol gene of HIV-1 and suggest that its activity may be controlled by a complex interplay of cis-regulatory elements.
Naturally occurring escape mutants of hepatitis B virus with various mutations in the S gene in carriers seropositive for antibody to hepatitis B surface antigen
Authors: Yamamoto K , Horikita M , Tsuda F , Itoh K , Akahane Y , Yotsumoto S , Okamoto H , Miyakawa Y , Mayumi M
Published: January 2020

Hepatitis B virus (HBV) DNA was extracted from sera of six carriers with hepatitis B e antigen as well as antibody to hepatitis B surface antigen and sequenced within the pre-S regions and the S gene. HBV DNA clones from five of these carriers had point mutations in the S gene, resulting in conversion from Ile-126 or Thr-126 of the wild-type virus to Ser-126 or Asn-126 in three carriers and conversion from Gly-145 to Arg-145 in three of them; clones with Asn-126 or Arg-145 were found in one carrier. All 12 clones from the other carrier had an insertion of 24 bp encoding an additional eight amino acids between Thr-123 and Cys-124. In addition, all or at least some of the HBV DNA clones from these carriers had in-phase deletions in the 5' terminus of the pre-S2 region. These results indicate that HBV escape mutants with mutations in the S gene affecting the expression of group-specific determinants would survive in some carriers after they seroconvert to antibody against surface antigen. Carriers with HBV escape mutants may transmit HBV either by donation of blood units without detectable surface antigen or through community-acquired infection, which would hardly be prevented by current hepatitis B immuneglobulin or vaccines.
The human immunodeficiency virus type 1 Tat protein transactivates tumor necrosis factor beta gene expression through a TAR-like structure
Authors: Buonaguro L , Buonaguro F M , Giraldo G , Ensoli B
Published: January 2020

We have previously shown that the Tat protein of human immunodeficiency virus type 1 (HIV-1) transactivates tumor necrosis factor alpha and beta (TNF alpha and TNF beta) gene expression in HIV-1-infected and in tat-transfected T-lymphocytic and monocytic cell lines. The product encoded by the first exon of the tat gene (amino acids 1 to 72) is sufficient for this transactivation. Here we show that (i) the NF-kappa B and Sp1 binding sites of the TNF beta promoter are required for Tat-mediated transactivation and (ii) a predicted stem-loop structure in the TNF beta mRNA leader region, which resembles the Tat-responsive element of the HIV-1 long terminal repeat (TAR) and which is therefore termed TAR-like, is essential for TNF beta transactivation by Tat. These data suggest that similar promoter regulatory elements are necessary for Tat-mediated transactivation of both TNF beta and HIV-1 gene expression. This represents the first demonstration of a cellular gene with a regulatory element downstream of the transcriptional initiation site that, like TAR, may function as an RNA element.
Antigenic specificities of human CD4+ T-cell clones recovered from recurrent genital herpes simplex virus type 2 lesions
Authors: Koelle D M , Corey L , Burke R L , Eisenberg R J , Cohen G H , Pichyangkura R , Triezenberg S J
Published: January 2020

Lesions resulting from recurrent genital herpes simplex virus (HSV) infection are characterized by infiltration of CD4+ lymphocytes. We have investigated the antigenic specificity of 47 HSV-specific CD4+ T-cell clones recovered from the HSV-2 buttock and thigh lesions of five patients. Clones with proliferative responses to recombinant truncated glycoprotein B (gB) or gD of HSV-2 or purified natural gC of HSV-2 comprised a minority of the total number of HSV-specific clones isolated from lesions. The gC2- and gD2-specific CD4+ clones had cytotoxic activity. The approximate locations of the HSV-2 genes encoding HSV-2 type-specific CD4+ antigens have been determined by using HSV-1 x HSV-2 intertypic recombinant virus and include the approximate map regions 0.30 to 0.46, 0.59 to 0.67, 0.67 to 0.73, and 0.82 to 1.0 units. The antigenic specificity of an HLA DQ2-restricted, HSV-2 type-specific T-cell clone was mapped to amino acids 425 to 444 of VP16 of HSV-2 by sequential use of an intertypic recombinant virus containing VP16 of HSV-2 in an HSV-1 background, recombinant VP16 fusion proteins, and synthetic peptides. Each of the remaining four patients also yielded at least one type-specific T-cell clone reactive with an HSV-2 epitope mapping to approximately 0.67 to 0.73 map units. The antigenic specificities of lesion-derived CD4+ T-cell clones are quite diverse and include at least 10 epitopes. Human T-cell clones reactive with gC and VP16 are reported here for the first time.
The UL21 gene products of herpes simplex virus 1 are dispensable for growth in cultured cells
Authors: Baines J D , Koyama A H , Huang T , Roizman B
Published: January 2020

A viral deletion mutant (delta UL21) that lacked the sequences encoding 484 of the predicted first 535 amino acids of the UL21 open reading frame was genetically engineered and studied with respect to its phenotype in cells in culture. We report the following. (i) The replication of delta UL21 was identical to that of the parent herpes simplex virus 1 (HSV-1) strain F in Vero cells, but the yields were three- to fivefold lower than those of the parent virus in human embryonic lung cells. (ii) To characterize the UL21 protein, we immunized rabbits against a purified bacterial fusion protein consisting of glutathione S-transferase fused to the majority of the coding domain of the UL21 gene. Rabbit antiserum directed against the fusion protein recognized a broad band with an apparent M(r) of 62,000 to 64,000 in lysates of cells infected with HSV-1 strain F and in virions purified from the infected cell cytoplasm. This band was absent from lysates of mock-infected cells or cells infected with the delta UL21 virus. The band was significantly reduced in intensity in lysates of cells infected in the presence of phosphonoacetic acid, indicating that it is expressed as a late (gamma 1) gene. (iii) Immunofluorescence studies localized the UL21 antigen primarily in brightly staining granules in the cytoplasms of infected cells. Taken together, the data indicate that the UL21 protein is a virion component dispensable for all aspects of replication of HSV-1 in the cells tested. The electrophoretic mobility of the UL21 protein suggests that it is extensively modified posttranslationally.
Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites
Authors: Burck P J , Berg D H , Luk T P , Sassmannshausen L M , Wakulchik M , Smith D P , Hsiung H M , Becker G W , Gibson W , Villarreal E C
Published: January 2020

The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein, subjected to the same activation conditions as the 28-kDa proteinase, had no enzymatic activity against the HCMV M- and R-site peptide substrates.
The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site
Authors: Staffa A , Cochrane A
Published: January 2020

Proportional expression of retroviral genes requires that splicing of the viral primary transcript be an inefficient process. Much of our current knowledge about retroviral suboptimal splicing comes from studies with Rous sarcoma virus. In this report, we describe the use of chimeric introns composed of human beta-globin and human immunodeficiency virus type 1 (HIV-1) splice sites to establish the basis for inefficient splicing of the intron which comprises most of the HIV-1 env coding sequences (referred to as the tat/rev intron). S1 RNA analysis of transfected COS-7 cells revealed that the 3' splice site (3' ss) of this region was significantly less efficient than the 3' ss of the first intron of beta-globin. Deletion of sequences flanking the tat/rev intron 3' ss demonstrated that the requirements for its inefficiency reside within the region that is expected to comprise the essential signals for splicing (i.e., the branchpoint region, the polypyrimidine tract, and the AG dinucleotide). Introduction of an exact copy of the efficient beta-globin branchpoint sequence within a highly conserved region rendered the tat/rev intron 3' ss highly efficient. Improvement of the polypyrimidine tract also increased the splicing efficiency, but to a degree slightly less than that obtained with the branchpoint mutation. Subsequent examination of the tat/rev intron 5' splice site in a heterologous context revealed that it is efficiently utilized. These results indicate that both a poor branchpoint region and a poor polypyrimidine tract are responsible for the low splicing efficiency of the HIV-1 tat/rev intron. It is of fundamental interest to establish the basis for inefficient splicing of the HIV-1 tat/rev intron since it may provide the key to understanding why nuclear export of mRNAs encoding HIV-1 structural proteins is Rev dependent.
Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus
Authors: Horimoto T , Kawaoka Y
Published: January 2020

To obtain direct evidence for a relationship between hemagglutinin (HA) cleavability and the virulence of avian influenza A viruses, we generated a series of HA cleavage mutants from a virulent virus, A/turkey/Ontario/7732/66 (H5N9), by reverse genetics. A transfectant virus containing the wild-type HA with R-R-R-K-K-R at the cleavage site, which was readily cleaved by endogenous proteases in chicken embryo fibroblasts (CEF), was highly virulent in intramuscularly or intranasally/orally inoculated chickens. By contrast, a mutant containing the HA with an avirulent-like sequence (R-E-T-R) at the cleavage site, which was not cleaved by the proteases in CEF, was avirulent in chickens, indicating that a genetic alteration confined to the HA cleavage site can affect cleavability and virulence. Mutant viruses with HA cleavage site sequences of T-R-R-K-K-R or T-T-R-K-K-R were as virulent as viruses with the wild-type HA, whereas a mutant with a two-amino-acid deletion but retention of four consecutive basic residues (R-K-K-R) was as avirulent as a virus with the avirulent-type HA. Interestingly, although a mutant containing an HA with R-R-R-K-T-R, which has reduced cleavability in CEF, was as virulent as viruses with high HA cleavability when given intramuscularly, it was less virulent when given intranasally/orally. We conclude that the degree of HA cleavability in CEF predicts the virulence of avian influenza viruses.
Assignment of disulfide bridges in the fusion glycoprotein of Sendai virus
Authors: Iwata S , Schmidt A C , Titani K , Suzuki M , Kido H , Gotoh B , Hamaguchi M , Nagai Y
Published: January 2020

The mature fusion (F) glycoprotein of the paramyxovirus family consists of two disulfide-linked subunits, the N-terminal F2 and the C-terminal F1 subunits, and contains 10 cysteine residues which are highly conserved at specific positions. The high level of conservation strongly suggests that they are indeed disulfide linked and play important roles in the folding and functioning of the molecule. However, it has not even been clarified which cysteine residues link the F2 and F1 subunits. This report describes our assignment of the disulfide bridges in purified Sendai virus F glycoprotein by fragmentation of the polypeptide and isolation of cystine-containing peptides and determination of their N-terminal sequences. The data demonstrate that all of the 10 cysteine residues participate in disulfide bridges and that Cys-70, the only cysteine in F2, and Cys-199, the most upstream cysteine in F1, form the interchain bond. Of the remaining eight cysteine residues clustered near the transmembrane domain of F1, the specific bridges identified are Cys-338 to Cys-347 and Cys-362 to Cys-370. Although no exact pairings between the subsequent four residues were defined, it seems likely that the most downstream, Cys-424, is linked to Cys-394, Cys-399, or Cys-401. Thus, we conclude that the cysteine-rich domain indeed contributes to the formation of a bunched structure containing at least two tandem cystine loops.
Uncoupled expression of Moloney murine leukemia virus envelope polypeptides SU and TM: a functional analysis of the role of TM domains in viral entry
Authors: Ragheb J A , Anderson W F
Published: January 2020

Moloney murine leukemia virus ecotropic envelope expression plasmids were used to demonstrate that the synthesis of the retroviral envelope SU and TM polypeptides can be uncoupled with retention of biologic activity. By substituting a glycosyl-phosphatidylinositol (GPI) membrane anchor for part or all of the retroviral envelope transmembrane protein and creating several deletion variants of the TM subunit, we have begun to dissect the role of the TM protein in envelope function. We show that a GPI-anchored envelope can be incorporated into virions and binds receptor. We found that the envelope cytoplasmic tail, while not required, influences the efficiency of retroviral transduction at some step after membrane fusion (possibly by interacting with core). The membrane-spanning domain of TM is involved in membrane fusion, and this function is distinct from its role as a membrane anchor. As few as eight amino acids of the putative membrane-spanning domain are sufficient to achieve membrane anchoring of envelope but not to mediate membrane fusion. In addition, though not required, the membrane-spanning domain may have some direct role in the incorporation of envelope into virions. Finally, the extracellular domain of TM, besides containing the putative fusion domain and interacting with SU, may influence the synthesis or stability and the glycosylation of envelope, possibly by affecting oligomerization of the complex and proper intracellular transit.
Dynamic behavior of hepatitis C virus quasispecies in patients undergoing orthotopic liver transplantation
Authors: Martell M , Esteban J I , Quer J , Vargas V , Esteban R , Guardia J , Gómez J
Published: January 2020

We have studied the distribution of viral sequences from the 5' noncoding region and from a fragment of the E2/NS2 region of the hepatitis C virus (HCV) genome in samples obtained before and after liver transplantation in two patients with HCV cirrhosis. The population of viral sequences in both regions were established by sequencing cloned PCR products. In both cases, the complexity of the viral quasispecies decreased after transplantation, although the consensus nucleotide and amino acid sequences remained unchanged. It is suggested that both positive and negative selection and random sampling events contribute substantially in shaping the genetic composition of HCV quasispecies and that recurrence of HCV infection may occur under equilibrium conditions.
Changes in RNA expression pattern during the malignant progression of cottontail rabbit papillomavirus-induced tumors in rabbits
Authors: Zeltner R , Borenstein L A , Wettstein F O , Iftner T
Published: January 2020

Cottontail rabbit papillomavirus induces strictly epithelial tumors in both cottontail and domestic rabbits. A high proportion of the initial benign papillomas progress within 8 to 14 months to invasive carcinomas. With the help of mRNA-specific riboprobes for E6, E7, E1, E2, L1 and L2, we investigated by in situ hybridization the RNA expression pattern of cottontail rabbit papillomavirus in tissue sections of biopsies from different stages of tumor development. Common features of all lesions were high levels of E6 and E7 mRNAs and low levels of E1 and E2 mRNAs. In agreement with earlier reports, there was no evidence for a major mRNA class equivalent to the prominent E1-E4 RNA of human papillomavirus types 6/11 and 16. In cottontail rabbit papillomas, high levels of E6 and E7 mRNAs were present in the upper differentiated epithelial layers. These layers also contained most of the E1 and E2 mRNAs and the viral DNA. In contrast, papillomas of domestic rabbits revealed the opposite differentiation-dependent expression pattern for the E6 and E7 mRNAs; there were strong signals in the basal layers, and these declined with increased differentiation. Transcripts encoding the L1 mRNA were detected only in a few isolated cells of the granular layer. There was no difference between the amounts of E6, E7, E1, and E2 mRNAs present in highly dysplastic tissue and those present in adjacent normal papillomatous epithelium within a progressing papilloma. However, late transcripts and viral DNA detectable only in the upper layers of the papilloma were present throughout the thickness of the dysplastic tissue, indicating a newly acquired permissiveness of the dysplastic cells for viral DNA replication and late transcription. Carcinomas in general had the same expression patterns for E6, E7, and E1 but were dissimilar in the levels of expression of E2 and late transcripts.
Cloning and expression of an equine herpesvirus 1 origin-binding protein
Authors: Martin D W , Deb S
Published: January 2020

Equine herpesvirus 1 (EHV-1) is an important pathogen of horses and is closely related to several important human pathogens, herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus. The EHV-1 genome contains open reading frames similar in sequence to the HSV-1 replication genes. PCR was used to clone EHV-1 gene 53, which is similar in sequence to the HSV-1 UL9 gene. The gene 53 product has regions of striking similarity to the HSV-1 UL9 and VZV gene 51 products. In vitro transcription and translation of this gene generated a protein of 87 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further characterization of this protein was accomplished through the use of gel shift analysis. The in vitro-synthesized protein bound sequence specifically to EHV-1 OriS as well as HSV-1 OriS. A site was used in gel shift analysis to show that the EHV-1 origin-binding protein bound to the same consensus site as the HSV-1 origin-binding protein, 5'-CGTTCGCACTT-3'. Using a nuclear extract of EHV-1-infected RK13 cells, we have identified an activity that interacts similarly with this consensus site. In gel shift assays, the retarded band arising from the nuclear extract migrated similarly to the retarded band arising from in vitro-translated EHV-1 gene 53. An N-terminal deletion of EHV-1 gene 53 was also created, expressed in vitro, and used in gel shift assays to localize the DNA-binding domain. Results of these experiments indicated that amino acids 1 to 499 were dispensable for binding and that the C-terminal fragment (amino acids 500 to 888) recognized the same consensus site as did the wild-type protein. Thus, the product of EHV-1 gene 53 is an origin-binding protein with a high degree of similarity to the HSV-1 and varicella-zoster virus origin-binding proteins and possibly serves as the initiator of DNA replication in EHV-1.