bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of potato virus X RNAs by membrane-containing extracts
Authors: Doronin S V , Hemenway C
Published: January 2020

Membrane-containing extracts isolated from tobacco plants infected with the plus-strand RNA virus, potato virus X (PVX), supported synthesis of four major, high-molecular-weight PVX RNA products (R1 to R4). Nuclease digestion and hybridization studies indicated that R1 and R2 are a mixture of partially single-stranded replicative intermediates and double-stranded replicative forms. R3 and R4 are double-stranded products containing sequences typical of the two major PVX subgenomic RNAs. The newly synthesized RNAs were demonstrated to have predominantly plus-strand polarity. Synthesis of these products was remarkably stable in the presence of ionic detergents.
The adenovirus E3-14.7K protein and the E3-10.4K/14.5K complex of proteins, which independently inhibit tumor necrosis factor (TNF)-induced apoptosis, also independently inhibit TNF-induced release of arachidonic acid
Authors: Krajcsi P , Dimitrov T , Hermiston T W , Tollefson A E , Ranheim T S , Vande Pol S B , Stephenson A H , Wold W S
Published: January 2020

Tumor necrosis factor (TNF) is an inflammatory cytokine that inhibits the replication of many viruses in cultured cells. We have reported that adenovirus (Ad) infection of TNF-resistant mouse cells renders them susceptible to lysis by TNF and that two sets of proteins encoded by the E3 transcription unit block TNF cytolysis. The E3 protein sets are named E3-14.7K (14,700 kDa) and E3-10.4K/14.5K (a complex of two proteins of 10,400 and 14,500 kDa). TNF activation of the 85-kDa cytosolic phospholipase A2 (cPLA2) is thought to be essential for TNF cytolysis (i.e.,TNF-induced apoptosis). Here we provide evidence that cPLA2 is important in the response of Ad-infected cells to TNF and that the mechanism by which E3-14.7K and E3-10.4K/14.5K inhibit TNF cytolysis is by inhibiting TNF activation of cPLA2. cPLA2 cleaves arachidonic acid (AA) specifically from membrane phospholipids; therefore, cPLA2 activity was measured by the release of 3H-AA from cells prelabeled with 3H-AA. Uninfected cells or cells infected with wild-type Ad were not lysed and did not release 3H-AA in response to TNF. In contrast, TNF treatment induced cytolysis and 3H-AA release in uninfected cells sensitized to TNF by treatment with cycloheximide and also in infected cells sensitized to TNF by expression of E1A. In C127 cells, in which either E3-14.7K or E3-10.4K/14.5K inhibits TNF cytolysis, either set of proteins inhibited TNF-induced release of 3H-AA. In C3HA cells, in which E3-14.7K but not E3-10.4K/14.5K prevents TNF cytolysis, E3-14.7K but not E3-10.4K/14.5K prevented TNF-induced release of 3H-AA. When five virus mutants with lesions in E3-14.7K were examined, there was a perfect correlation between a mutant's ability to inhibit both TNF-induced cytolysis and release of 3H-AA. E3-14.7K expressed in two stably transfected C127 cell lines prevented both TNF-cycloheximide-induced cytolysis and release of 3H-AA. The E3 proteins also prevented TNF-induced cytolysis and release of 3H-AA in mouse L929 cells, which are spontaneously sensitive to TNF. TNF cytolysis was blocked by dexamethasone, an inhibitor of PLA2 activity, and by nordihydroquaiaretic acid, which inhibits the metabolism of AA to the leukotrienes. Indomethacin, which blocks the formation of prostaglandins from AA, did not inhibit TNF cytolysis. The leukotrienes and prostaglandins are amplifiers of the inflammatory response. We propose that E3-14.7K and E3-10.4K/14.5K function independently in Ad infection to inhibit both cytolysis and inflammation induced by TNF.
AP1 enhances polyomavirus DNA replication by promoting T-antigen-mediated unwinding of DNA
Authors: Guo W , Tang W J , Bu X , Bermudez V , Martin M , Folk W R
Published: January 2020

An early step in the initiation of polyomavirus DNA replication is viral large-T-antigen-mediated unwinding of the origin. We report that components of the AP1 transcription factor, Fos and Jun, interact with T antigen in vitro to enhance unwinding of the viral origin. This provides a biochemical basis for the capacity of AP1 to activate viral DNA replication in vivo.
RNA replication by a respiratory syncytial virus RNA analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end
Authors: Samal S K , Collins P L
Published: January 2020

Genome analogs ("minigenomes") of Sendai and measles viruses replicate efficiently only if their nucleotide length is an even multiple of six, a requirement called the rule of six (P. Calain and L. Roux, J. Virol. 67:4822-4830, 1993; M. S. Sidhu, J. Chan, K. Kaelin, P. Spielhofer, F. Radecke, H. Schneider, M. Masurekar, P. C. Dowling, M. A. Billeter, and S. A. Udem, Virology 208:800-807, 1995). The existence of a comparable requirement was tested for respiratory syncytial virus (RSV), which also is a member of family Paramyxoviridae and whose natural genome length also is a multiple of six. An internally truncated analog of RSV positive-sense replicative intermediate RNA (antigenome) bearing the chloramphenicol acetyltransferase gene as a reporter was synthesized from cDNA in vitro. This RNA was transfected into cells which were infected with RSV as a helper. Miniantigenomic RNA was indistinguishable from previously studied negative-sense minigenome RNA in its ability to participate in transcription, RNA replication, and incorporation into transmissible particles. Sixteen miniantigenomes which were of slightly different lengths and which in aggregate represented multiples of a wide range of integers including 1 to 15 were constructed. During transfection and two serial passages, the various miniantigenomes were essentially indistinguishable with regard to the efficiency of transcription, RNA replication, and packaging into transmissible particles. Progeny minigenomes of six different mutants were recovered postpassage, copied into cDNA, cloned, and sequenced completely. The length of each of these RNAs was found to have remained unchanged during replication and passage. Thus, RSV transcription and replication appear to lack the requirement that the template length be an even multiple of an integer such as six, which for Sendai and measles viruses is obligatory for nucleocapsid function. Each of the in vitro-synthesized miniantigenomes used in transfection contained a nonviral extension of three nucleotides, GGG, on the 5' (leader) end contributed by the T7 promoter. The termini of the recovered minigenomes were examined for five mutants by RNA circularization followed by cDNA synthesis, amplification, cloning, and sequencing. Unexpectedly, each recovered minigenome contained the complement of this nonviral extension on the 3' (leader) end, showing that it had been faithfully copied and maintained during RNA replication and passage. The nonviral trinucleotide did not appear to affect the activity of the template.
Chronic human immunodeficiency virus type 1 infection of myeloid cells disrupts the autoregulatory control of the NF-kappaB/Rel pathway via enhanced IkappaBalpha degradation
Authors: DeLuca C , Roulston A , Koromilas A , Wainberg M A , Hiscott J
Published: January 2020

Productive human immunodeficiency virus type 1 (HIV-1) infection causes sustained NF-kappaB DNA-binding activity in chronically infected monocytic cells. A direct temporal correlation exists between HIV infection and the appearance of NF-kappaB DNA-binding activity in myelomonoblastic PLB-985 cells. To examine the molecular basis of constitutive NF-kappaB DNA-binding activity in HIV1 -infected cells, we analyzed the phosphorylation and turnover of IkappaBalpha protein, the activity of the double-stranded RNA-dependent protein kinase (PKR) and the intracellular levels of NF-kappaB subunits in the PLB-985 and U937 myeloid cell models. HIV-1 infection resulted in constitutive, low-level expression of type 1 interferon (IFN) at the mRNA level. Constitutive PKR activity was also detected in HIV-1-infected cells as a result of low-level IFN production, since the addition of anti-IFN-alpha/beta antibody to the cells decreased PKR expression. Furthermore, the analysis of IkappaBalpha turnover demonstrated an increased degradation of IkappaBalpha in HIV-1-infected cells that may account for the constitutive DNA binding activity. A dramatic increase in the intracellular levels of NF-kappaB subunits c-Rel and NF-kappaB2 p100 and a moderate increase in NF-kappaB2 p52 and RelA(p65) were detected in HIV-1-infected cells, whereas NF-kappaB1 p105/p50 levels were not altered relative to the levels in uninfected cells. We suggest that HIV-1 infection of myeloid cells induces IFN production and PKR activity, which in turn contribute to enhanced IkappaBalpha phosphorylation and subsequent degradation. Nuclear translocation of NF-kappaB subunits may ultimately increase the intracellular pool of NF-kappaB/IkappaBalpha by an autoregulatory mechanism. Enhanced turnover of IkappaBalpha and the accumulation of NF-kappaB/Rel proteins may contribute to the chronically activated state of HIV-1-infected cells.
Mechanisms of human immunodeficiency virus Type 1 (HIV-1) neutralization: irreversible inactivation of infectivity by anti-HIV-1 antibody
Authors: McDougal J S , Kennedy M S , Orloff S L , Nicholson J K , Spira T J
Published: January 2020

An assay for the neutralization of human immunodeficiency virus type 1 (HIV-1) is described in which the reduction in infectious titer of HIV-1 after preincubation at 37 degrees C with antibody-positive serum is the measure of neutralization. The assay format and its controls allow several experimental manipulations that, taken together, indicate an effect of antibody on HIV-1 infectivity that occurs before or independently of HIV-1 attachment. The direct inactivation of HIV-1 infectivity by antibody is irreversible and temperature dependent, requires a bivalent antibody directed against accessible envelope determinants, and does not require a heat-labile or (Ca2+)- or (Mg2+)-dependent cofactor. The mechanism of inactivation cannot be explained by agglutination of virus, nor is it associated with disruption or dissociation of envelope protein from virions. Rather, the antibody is likely to perturb some metastable property of the envelope that is required for entry. Laboratory-adapted HIV-1 isolates were more sensitive to the inactivating effects of sera than were primary patient isolates. The latter were particularly resistant to inactivation by contemporary autologous sera, a feature not explained by blocking antibodies. Additional studies showed a weak relationship between disease course and serum inactivation of the reference LAI laboratory strain of HIV-1. Heteroduplex analysis and autologous inactivation assays of sequential specimens from individual patients indicate that over time, the viral quasispecies that emerge and dominate are resistant to the inactivating effects of earlier sera.
The variable 3' ends of a human cytomegalovirus oriLyt transcript (SRT) overlap an essential, conserved replicator element
Authors: Huang L , Zhu Y , Anders D G
Published: January 2020

The genetically defined human cytomegalovirus (HCMV) lytic-phase replicator, oriLyt, comprises more than 2 kb in a structurally complex region that spans a variety of potential transcription control signals. Several transcripts originate within or cross oriLyt, and we are studying these oriLyt transcription units to determine whether they participate in initiating or regulating lytic-phase DNA synthesis. Results presented here establish the temporal accumulation and structure of the smallest replicator transcript, which we call SRT, and identify a single-sequence element essential to replicator function. SRT was detected as early as 2 h after HCMV infection of human fibroblast cells; transcript levels increased by 24 h and continued to increase thereafter. Consistent with its early appearance, treatment of HCMV-infected cells with the viral DNA polymerase inhibitor phosphonoformic acid had no effect on SRT accumulation; however, no SRT was detected in RNA preparations from cycloheximide-treated infected cells. Additional Northern (RNA) analysis localized the 0.2- to 0.25-kb SRT to an apparently noncoding segment near the center of the oriLyt core region. Reverse transcriptase PCR (rapid amplification of cDNA 5' ends [5'-RACE]) identified a single 5' end. In transient-transfection assays, the sequence immediately upstream of SRT functioned as a promoter responsive to HCMV infection when placed upstream of a reporter gene, suggesting that SRT is the product of a discrete transcription unit. RNA ligase-mediated 3'-RACE showed that SRT is not polyadenylated and has heterogeneous 3' ends within a roughly 45-nucleotide window overlapping an oligopyrimidine sequence having counterparts in the lytic-phase replicators of several herpesviruses. Mutation of the oligopyrimidine element showed that it is essential to oriLyt replicator function; it is the only essential single-sequence HCMV oriLyt replicator element described to date. Collectively, the location of SRT near the center of the oriLyt core region, its early expression, its overlapping relationship with a sequence element essential to replicator function, and its similarities to replicator transcripts in other systems suggest the possibility that SRT plays a role in initiating or regulating HCMV lytic-phase DNA synthesis.
The human immunodeficiency virus type 1 Vif protein modulates the postpenetration stability of viral nucleoprotein complexes
Authors: Simon J H , Malim M H
Published: January 2020

The vif gene of human immunodeficiency virus type 1 is absolutely required for productive infection of primary cells derived from human blood and certain immortalized T lymphoid cells, for example, H9. Cells with this restrictive phenotype are termed nonpermissive, whereas cell lines in which vif-deficient virus can replicate efficiently are known as permissive. In this paper, we describe experiments in which virus stocks derived from single-cycle infections of strictly nonpermissive H9 cells were used to determine the fate of vif-deficient infections. By PCR-based approaches, it was found that Vif has no significant impact on the biosynthetic capability of the virion reverse transcriptase in infected C8166 T cells. Specifically, the initial appearance of all DNA species up to and including initiated second (plus) strands as well as the early accumulation of these replicative intermediates is equivalent for wild-type and vif-deficient infections. However, whereas these viral DNAs are stably maintained in wild-type infections and can proceed to establish proviruses, they are largely degraded by the later time points of vif-deficient infections and, as a result, are prevented from forming proviruses. Subcellular fractionation analyses indicated that the majority of viral DNA is localized to the nucleus within 2 h of infection and that the turnover of reverse transcripts that occurs in these vif-deficient infections presumably takes place in the nucleus. Given that the ultimate infection phenotype of the virions is determined during virus production, we propose that Vif is required for an aspect of virus assembly and/or maturation that endows penetrating viral nucleoprotein cores with the ability to mature into functional preintegration complexes that can proceed to provirus establishment. In contrast, viruses that are produced in the absence of Vif give rise to nucleoprotein complexes that disassemble prematurely in challenged cells and fail to protect their RNA/DNA contents from nucleolytic destruction.
Characterization of the functional properties of env genes from long-term survivors of human immunodeficiency virus type 1 infection
Authors: Connor R I , Sheridan K E , Lai C , Zhang L , Ho D D
Published: January 2020

A small number of persons infected with human immunodeficiency virus type 1 (HIV-1) remain clinically and immunologically healthy for more than a decade after infection. Recent reports suggest that these individuals may be infected with an attenuated strain of HIV-1; however, a common genetic basis for viral attenuation has not been found in all cases. In the present study, we examined the functional properties of the HIV-1 env genes from six long-term survivors. env clones were generated by PCR amplification of proviral env sequences, followed by cloning of the amplified regions into expression vectors. Eight to ten clones from each subject were screened by transient transfection for expression of the envelope precursor glycoprotein, gp160. Those clones expressing gp160 were then cotransfected with an HIV-1 luciferase reporter vector, pNL4-3Env(-)LUC(+) and evaluated for their ability to mediate infection of phytohemagglutinin-activated peripheral blood mononuclear cells in single-cycle infectivity assays. Clones expressing gp160 were identified for all six long-term survivors, indicating the presence of proviral env genes with intact open reading frames. For two subjects, D and DH, the encoded envelope glycoproteins yielded high levels of luciferase activity when pseudotyped onto HIV-1 virions and tested in single-cycle infectivity assays. In contrast, envelope glycoproteins cloned from four other long-term survivors were poorly processed and failed to mediate infection. Sequencing of the gp120/41 cleavage site and conserved gp41 cysteine residues of these clones did not reveal any obvious mutations to explain the functional defects. The functional activity of env clones from long-term survivors D and DH was comparable to that seen with several primary HIV-1 env genes cloned from individuals with disease progression and AIDS. These results suggest that the long-term survival of subjects D and DH is not associated with overt functional defects in env; however, functional abnormalities in env may contribute to maintaining a long-term asymptomatic state in the other four cases we studied.
Lack of effect of mouse adenovirus type 1 infection on cell surface expression of major histocompatibility complex class I antigens
Authors: Kring S C , Spindler K R
Published: January 2020

It has been proposed that adenoviruses establish and maintain persistent infections by reducing the class I major histocompatibility complex-associated presentation of viral antigens to cytotoxic T lymphocytes, leading to ineffective cell-mediated immunity and impaired clearance of infected cells (W.S.M. Wold and L. R. Gooding, Virology 184:1-8, 1991). Early region 3 of human adenovirus types 2 and 5 encodes a 19-kDa glycoprotein that associates with the class I major histocompatibility complex (MHC) antigens in the endoplasmic reticulum and prevents their maturation and transport to the cell surface. Early region 1A of human adenovirus type 12 encodes a protein that inhibits class I MHC mRNA production at the transcriptional or posttranscriptional processing level. Unlike human adenovirus infections, however, mouse adenovirus type 1 (MAV-1) infection of a variety of cell types did not affect the surface expression of 10 different mouse class I MHC allotypes. MAV-1-infected cells also regenerated cell surface class I MHC antigens following proteolytic removal as efficiently as mock-infected cells. The ability of cells to present antigen to class I MHC (Kb)-ovalbumin-specific T-cell hybridoma cells was likewise unaltered by MAV-1 infection. Thus, the ability of MAV-1 to persist cannot be explained by the model of reduced class I MHC-associated antigen presentation proposed for human adenoviruses.
Endogenous reverse transcription assays reveal high-level resistance to the triphosphate of (-)2'-dideoxy-3'-thiacytidine by mutated M184V human immunodeficiency virus type 1
Authors: Quan Y , Gu Z , Li X , Li Z , Morrow C D , Wainberg M A
Published: January 2020

Kinetic analysis showed that the Ki values and the Ki/Km ratios for mutated, recombinant M184V human immunodeficiency virus type 1 reverse transcriptase (RT) for (-)2'-dideoxy-3'-thiacytidine triphosphate (3TCTP) were 35-fold higher than the equivalent values for wild-type RT but only about twice as high as the equivalent values for each of the triphosphates of ddC (ddCTP) and ddA (ddATP). Fully endogenous RT assays showed that viruses containing the M184V substitution were highly resistant to 3TCTP, with an increase in the 50% inhibitory concentration of 250-fold in comparison with wild-type recombinant virus.
The human immunodeficiency virus type 1 capsid p2 domain confers sensitivity to the cyclophilin-binding drug SDZ NIM 811
Authors: Dorfman T , Göttlinger H G
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) specifically incorporates the host cell peptidyl-prolyl isomerase cyclophilin A into virions via contacts with the capsid (CA) domain of the Gag polyprotein Pr55gag. The immunosuppressant drug cyclosporin A and the nonimmunosuppressive cyclosporin A analog SDZ NIM 811 bind to cyclophilin A and inhibit its incorporation into HIV-1 virions. Both drugs inhibit the virion association of cyclophilin A and the replication of HIV-1 with a similar dose dependence. In contrast, these compounds are inactive against other primate lentiviruses which do not incorporate cyclophilin A, such as simian immunodeficiency virus (SIV). To locate determinants which confer sensitivity to SDZ NIM 811, we generated chimeric proviruses between HIV-1 and SIVmac. A hybrid SIVmac which has the CA-p2 domain of the Gag polyprotein replaced by the corresponding domain from HIV-1 replicated in an established CD4+ cell line and in human but not macaque peripheral blood mononuclear cells. The transfer of the HIV-1 CA-p2 domain to SIVmac led to the efficient incorporation of cyclophilin A, and SDZ NIM 811 effectively inhibited both the virion association of cyclophilin A and the spread of the hybrid virus in infected cultures. We conclude that the HIV-1 CA-p2 domain contains determinants which confer the necessity to interact with cyclophilin A for efficient virus replication. Furthermore, our data show that the CA-p2 domain can play a crucial role in species tropism.
Epitopes exposed on hepatitis delta virus ribonucleoproteins
Authors: Bichko V V , Lemon S M , Wang J G , Hwang S , Lai M M , Taylor J M
Published: January 2020

A total of 17 antibodies, raised in several nonhuman species and specific for different regions on the delta antigen (delta Ag), were used to map, via immunoprecipitation, those domains exposed on the surface of the viral ribonucleoprotein (RNP). These studies showed that the domains for the nuclear localization signal and the C-terminal extension, unique to the large form of delta Ag, are exposed. Also exposed is the C-terminal region of the small form of delta Ag. In contrast, reactivity was not found with the coiled-coil domain needed for protein dimerization. When the hepatitis delta virus (HDV) RNA was released by treatment of viral RNP with vanadyl ribonucleoside complexes, no change in the pattern of delta Ag epitope presentation was detected, consistent with the interpretation that a multimeric protein structure persists in the absence of RNA. These RNP studies have implications not only for understanding of the process of HDV assembly but also for evaluation of the immune responses of an infected host to HDV replication.
Analysis of early region 3 mutants of mouse adenovirus type 1
Authors: Beard C W , Spindler K R
Published: January 2020

Early region 3 (E3) of mouse adenovirus type 1 has the potential to produce three proteins which have identical amino termini but unique carboxy-terminal sequences. Three recombinant deletion viruses were constructed so that each could produce only one of the three E3 proteins. A fourth mutant that should produce no E3 proteins was also constructed. These recombinants were able to grow in mouse 3T6 cells and produced wild-type levels of viral mRNAs and proteins except for those specifically deleted by the mutations. Early mRNA production from the mutant viruses was analyzed by reverse transcriptase PCR and confirmed that each deletion mutant would be able to produce only one of the three E3 proteins. Late mRNA production was analyzed by Northern (RNA) blotting and found to be similar in wild-type and mutant viruses. Capsid morphology was unaltered in the mutant viruses as seen by electron microscopy. Immunoprecipitation of E3 proteins from infections of mouse 3T6 cells using an antiserum specific for all three E3 proteins was used to examine the effect of the introduced mutations on protein expression. Two mutants produced only one class of E3 protein as predicted from their specific mutations and mRNA expression profiles. One mutant virus failed to produce any detectable E3 proteins. The predicted E3-null mutant was found to be leaky and could produce low levels of E3 proteins. Outbred Swiss mice were infected with the E3 mutant viruses to determine if the E3 proteins have an effect on the pathogenicity of the virus in mice. All of the mutants showed decreased pathogenicity as determined by increased 50% lethal doses, indicating that the proteins of the E3 region are important determinants of the pathogenesis of mouse adenovirus in its natural host.
Local and distant sequences are required for efficient readthrough of the barley yellow dwarf virus PAV coat protein gene stop codon
Authors: Brown C M , Dinesh-Kumar S P , Miller W A
Published: January 2020

Many viruses use stop codon readthrough as a strategy to produce extended coat or replicase proteins. The stop codon of the barley yellow dwarf virus (PAV serotype) coat protein gene is read through at a low rate. This produces an extended polypeptide which becomes part of the virion. We have analyzed the cis-acting sequences in the barley yellow dwarf virus PAV genome required for this programmed readthrough in vitro in wheat germ extracts and reticulocyte lysates and in vivo in oat protoplasts. Two regions 3' to the stop codon were required. Deletion of sections containing the first 5 of the 16 CCN NNN repeats located 3' of the stop codon greatly reduced readthrough in vitro and in vivo. Surprisingly, readthrough also required a second, more distal element that is located 697 to 758 bases 3' of the stop codon within the readthrough open reading frame. This element also functioned in vivo in oat protoplasts when placed more than 2 kb from the coat protein gene stop in the untranslated region following a GUS reporter gene. This is the first report of a long-range readthrough signal in viruses.
Cleavage site mutations in the encephalomyocarditis virus P3 region lethally abrogate the normal processing cascade
Authors: Hall D J , Palmenberg A C
Published: January 2020

Site-specific mutations within the proteinase 3C-dependent P3 region cleavage sequences of encephalomyocarditis virus have been constructed. The mutations altered the normal QG cleavage site dipeptide pairs of the 2C/3A, 3A/3B, 3B/3C, and 3C/3D junctions into QV, QC, QF, QY, and RG sequences. When translated in vitro in the context of full-length viral polyproteins, all mutations blocked endogenous 3C-mediated processing at their engineered sites and produced stable forms of the expected viral P3 precursors that were also resistant to cleavage by exogenously added recombinant 3C. Relative to wild-type viral sequences, each mutant form of P3 had a somewhat different ability to mediate overall polyprotein processing. Mutations at the 2C/3A, 3A/3B, and 3B/3C sites, for example, were generally less impaired than 3C/3D mutations, when the cleavage reactions were quantitated with cotranslated L-P1-2A precursors. A notable exception was mutant 3B3C(QG-->RG), which proved far less active than sibling mutants 3B3C(QG-->QF) and 3B3C(QG-->QV), a finding that possibly implicates this segment in the proper folding of an active 3C. When transfected into HeLa cells, all mutant sequences were lethal, presumably because of the reduced L-P1-2A processing levels or reduced RNA synthesis capacity. However, when specifically tested for the latter activity, all mutations except those at the 3C/3D cleavage site were indeed able to initiate and perpetuate viral RNA replication in transfected cells, albeit to RNA accumulation levels lower than those produced by wild-type sequences. The transfection effects could be mimicked with cell-free synthesized proteins, in that translation samples containing locked 3CD polymerase precursors were catalytically inactive in poly(A)-oligo(U)-dependent assays, while all other mutant processing samples initiated detectable RNA synthesis. Surprisingly, not only did the 3B/3C mutant sequences prove capable of directing RNA synthesis, but the viral RNA thus synthesized could be immunolabeled and precipitated with 3C-specific monoclonal antibody reagents, indicating an unexpected covalent attachment of the proteinase to the RNA product whenever this cleavage site was blocked.
Evidence that two latency-associated transcripts of herpes simplex virus type 1 are nonlinear
Authors: Wu T T , Su Y H , Block T M , Taylor J M
Published: January 2020

The latency-associated transcripts (LATs) of herpes simplex virus type 1 (HSV-1) are the only viral gene products that accumulate to abundant levels in latently infected cells. Others have reported species of 2.0, 1.50, and 1.45 kb; only the 2.0-kb species is seen in productively infected cells, and there is evidence that it behaves as an intron. We examined the LATs both in trigeminal ganglia of latently infected mice and in productively infected cultures of monkey CV-1 cells. After glyoxalation, RNA was subjected to high-resolution agarose gel electrophoresis and Northern (RNA) analysis, a procedure capable of resolving linear and nonlinear RNA species. Under these conditions, we resolved the 2.0-kb LAT into two species; the slower species was much more abundant and had a mobility significantly slower than expected for a linear RNA. To test the hypothesis that this RNA was in fact nonlinear, we used partial hydrolysis by sodium carbonate and oligonucleotide-directed RNase H digestion. These procedures changed the mobility of the slower species into that of the faster species. Similarly, the mobility of the 1.50-kb LAT, which was much more abundant than the 1.45-kb LAT, was changed by these procedures to that of the 1.45-kb LAT. Our data show that the two major LAT species are nonlinear, and they support an interpretation of stable lariat structures.
Determination and analysis of the complete nucleotide sequence of human herpesvirus
Authors: Nicholas J
Published: January 2020

Human herpesvirus 7 (HHV-7) is a recently isolated betaherpesvirus that is prevalent in the human population, with primary infection usually occurring in early childhood. HHV-7 is related to human herpesvirus 6 (HHV-6) in terms of both biological and, from limited prior DNA sequence analysis, genetic criteria. However, extensive analysis of the HHV-7 genome has not been reported, and the precise phylogenetic relationship of HHV-7 to the other human betaherpesviruses HHV-6 and human cytomegalovirus has not been determined. Here I report on the determination and analysis of the complete DNA sequence of HHV-7 strain JI. The data establish that the close biological relationship of HHV-6 and HHV-7 is reflected at the genetic level, where there is a very high degree of conservation of genetic content and encoded amino acid sequences. The data also delineate loci of divergence between the HHV-6 and HHV-7 genomes, which occur at the genome terminal in the region of the terminal direct-repeat elements and within limited regions of the unique component. Of potential significance with respect to biological and evolutionary divergence of HHV-6 and HHV-7 are notable structural differences in putative transcriptional regulatory genes specified by the direct-repeat and immediate-early region A loci of these viruses and the absence of an equivalent of the HHV-6 adeno-associated virus type 2 rep gene homolog in HHV-7.
A longitudinal study of feline immunodeficiency virus-specific cytotoxic T lymphocytes in experimentally infected cats, using antigen-specific induction
Authors: Beatty J A , Willett B J , Gault E A , Jarrett O
Published: January 2020

The evolution of the virus-specific cytotoxic T-lymphocyte response in two cats experimentally infected with feline immunodeficiency virus (FIV) was monitored. Effector cells were derived from peripheral blood lymphocytes during the acute and chronic phases of infection (0 to 21 and 62 to 127 weeks, respectively) and from the spleen and lymph nodes at 127 weeks after infection. Lymphocytes were restimulated in vitro with paraformaldehyde-fixed, autologous lymphoblasts which had been infected with recombinant vaccinia viruses expressing FIV GAG or ENV proteins. Unstimulated lymphocytes were also used as effectors in some assays. 51Cr-labelled autologous skin fibroblasts infected with recombinant vaccinia viruses were used as targets. FIV GAG-specific cytotoxic precursors were detected in restimulated circulating lymphocytes during acute infection in both cats. The onset of this activity was as early as 2 weeks postinfection (p.i.) in one cat. From 62 weeks p.i. neither FIV GAG- nor ENV-specific precursors could be detected in the peripheral blood. However, at 127 weeks p.i., GAG- and ENV-specific cytotoxic precursors were detected in lymphocytes isolated from lymph nodes. The FIV-specific cytotoxic cells were predominantly major histocompatibility complex class I restricted. No cytotoxic activity was detected from unstimulated lymphocytes. These studies demonstrate the use of an assay system for dissecting the FIV-specific cytotoxic cell response and show that precursor cells appear in the circulation very early after infection and prior to a detectable antibody response. Our results also suggest that the persistent high-level circulating antiviral cytotoxic T-lymphocyte responses seen in human immunodeficiency virus-infected humans may not be a feature of FIV infections in cats.
Insulin-like growth factor II blocks apoptosis of N-myc2-expressing woodchuck liver epithelial cells
Authors: Yang D , Faris R , Hixson D , Affigne S , Rogler C E
Published: January 2020

N-myc2 and insulin-like growth factor II (IGF-II) are coordinately overexpressed in the great majority of altered hepatic foci, which are the earliest precancerous lesions observed in the liver of woodchuck hepatitis virus carrier woodchucks, and these genes continue to be overexpressed in hepatocellular carcinomas (HCCs). We have investigated the function of these genes in woodchuck hepatocarcinogenesis by using a woodchuck liver epithelial cell line (WC-3). WC-3 cells react positively with a monoclonal antibody (12.8.5) against woodchuck oval cells, suggesting a lineage relationship with oval cells. Overexpression of N-myc2 in three WC-3 cell lines caused their morphological transformation and increased their growth rate and saturation density in medium containing 10% serum. Removal of serum from the medium increased cell death of the N-myc2-expressing lines, whereas cell death in control lines was minimal. The death of N-myc2-expressing WC-3 cells was accompanied by nucleosomal fragmentation of cellular DNA, and DAPI (4',6-diamidino-2-phenylindole) staining revealed condensation and fragmentation of the nuclei, suggesting that N-myc2-expressing WC-3 cells undergo apoptosis in the absence of serum. In colony regression assays, conducted in the absence of serum, control colonies were stable, while N-myc2-expressing colonies regressed to various degrees. Addition of recombinant human IGF-II to the serum-free medium blocked both cell death and colony regression in all the N-myc2-expressing lines. Therefore, coordinate overexpression of N-myc2 and IGF-II in woodchuck altered hepatic foci may allow cells which otherwise might die to survive and progress to hepatocellular carcinoma.
Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA
Authors: Shi P Y , Li W , Brinton M A
Published: January 2020

The first 96 nucleotides of the 5'noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3' SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3' SL RNA as the probe. Three RNA-protein complexes (complexes 1,2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3' SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3' SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.
Human immunodeficiency virus type 1 membrane fusion mediated by a laboratory-adapted strain and a primary isolate analyzed by resonance energy transfer
Authors: Litwin V , Nagashima K A , Ryder A M , Chang C H , Carver J M , Olson W C , Alizon M , Hasel K W , Maddon P J , Allaway G P
Published: January 2020

Previous studies of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein-mediated membrane fusion have focused on laboratory-adapted T-lymphotropic strains of the virus. The goal of this study was to characterize membrane fusion mediated by a primary HIV-1 isolate in comparison with a laboratory-adapted strain. To this end, a new fusion assay was developed on the basis of the principle of resonance energy transfer, using HeLa cells stably transfected with gp120/gp41 from the T-lymphotropic isolate HIV-1LA1 or the macrophage-tropic primary isolate HIV-1JR-FL. These cells fused with CD4+ target cell lines with a tropism mirroring that of infection by the two viruses. Of particular note, HeLa cells expressing HIV-1JR-FL gp120/gp41 fused only with PM1 cells, a clonal derivative of HUT 78, and not with other T-cell or macrophage cell lines. These results demonstrate that the envelope glycoproteins of these strains play a major role in mediating viral tropism. Despite significant differences exhibited by HIV-1JR-FL and HIV-1LAI in terms of tropism and sensitivity to neutralization by CD4-based proteins, the present study found that membrane fusion mediated by the envelope glycoproteins of these viruses had remarkably similar properties. In particular, the degree and kinetics of membrane fusion were similar, fusion occurred at neutral pH and was dependent on the presence of divalent cations. Inhibition of HIV-1JR-FL envelope glycoprotein-mediated membrane fusion by soluble CD4 and CD4-IgG2 occurred at concentrations similar to those required to neutralize this virus. Interestingly, higher concentrations of these agents were required to inhibit HIV-1LAI envelope glycoprotein-mediated membrane fusion, in contrast to the greater sensitivity of HIV-1LAI virions to neutralization by soluble CD4 and CD4-IgG2. This finding suggests that the mechanisms of fusion inhibition and neutralization of HIV-1 are distinct.
Detection of herpes simplex virus type 1 latency-associated transcript expression in trigeminal ganglia by in situ reverse transcriptase PCR
Authors: Ramakrishnan R , Poliani P L , Levine M , Glorioso J C , Fink D J
Published: January 2020

One of the defining characteristics of herpes simplex virus type 1 (HSV-1) infection is the ability of the virus to establish a lifelong latent state in neurons. We previously demonstrated (R. Ramakrishnan, A.J. Fink, G. Jiang, P. Desai, J. C. Glorioso, and M. Levine, J. Virol. 68:1864-1873, 1994) by in situ PCR that many more neurons contain viral genomes than are detected by in situ hybridization for HSV latency-associated transcripts (LATs). To determine whether all cells which contain genomes express LATs, we examined trigeminal ganglia for LATs 1 and 8 weeks after corneal scarification with ribonucleotide reductase-deficient HSV-1 by in situ reverse transcriptase PCR. The number of LAT-positive cells detected by in situ reverse transcriptase was substantially greater than the number of cells positive by in situ hybridization and appeared to be similar to the number of cells containing HSV genomes by in situ PCR and the number of ganglionic neurons that project to the cornea as detected by retrograde labeling with Fluorogold. These results demonstrate LAT expression in many neurons containing HSV-1 genomes.
Serine protein kinase activity associated with rotavirus phosphoprotein NSP5
Authors: Blackhall J , Fuentes A , Hansen K , Magnusson G
Published: January 2020

The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine. We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11. Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity. After metabolic labeling of NSP5 with 32Pi, only serine residues were phosphorylated. Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products. Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified. Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa. Moreover, there were polypeptides migrating between 28 and 35 kDa. Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28- to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation. Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A. The immunoprecipitated NSP5 possessed protein kinase activity. Incubation with [gamma-32P]ATP resulted in 32P labeling of 28- to 35-kDa NSP5. The distribution of 32P radioactivity between the components of the complex was similar to the phosphorylation in vivo. Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.
A novel human T-leukemia virus type 1 cell-to-cell transmission assay permits definition of SU glycoprotein amino acids important for infectivity
Authors: Delamarre L , Rosenberg A R , Pique C , Pham D , Dokhélar M C
Published: January 2020

Human T-leukemia virus type 1 (HTLV-1) envelope glycoproteins play a major role in viral transmission, which in the case of this virus occurs almost exclusively via cell-to-cell contact. Until very recently, the lack of an HTLV-1 infectivity assay precluded the determination of the HTLV-1 protein domains required for infectivity. Here, we describe an assay which allows the quantitative evaluation of HTLV-1 cell-to-cell transmission in a single round of infection. Using this assay, we demonstrate that in this system, cell-to-cell transmission is at least 100 times more efficient than transmission with free viral particles. We have examined 46 surface (SU) glycoprotein mutants in order to define the amino acids of the HTLV-1 SU glycoprotein required for full infectivity. We demonstrate that these amino acids are distributed along the entire length of the SU glycoprotein, including the N-terminus and C-terminus regions, which have not been previously defined as being important for HTLV-1 glycoprotein function. For most of the mutated glycoproteins, the capacity to mediate cell-to-cell transmission is correlated with the ability to induce formation of syncytia. This result indicates that the fusion capacity is the main factor responsible for infectivity mediated by the HTLV-1 SU envelope glycoprotein, as is the case for other retroviral glycoproteins. However, other factors must also intervene, since two of the mutated glycoproteins were correctly fusogenic but could not mediate cell-to-cell transmission. Existence of this phenotype shows that capacity for fusion is not sufficient to confer infectivity, even in cell-to-cell transmission, and could suggest that postfusion events involve the SU.
Human endogenous retrovirus type I-related viruses have an apparently widespread distribution within vertebrates
Authors: Martin J , Herniou E , Cook J , Waugh O'Neill R , Tristem M
Published: January 2020

Retroviruses from lower vertebrate hosts have been poorly characterized to date. Few sequences have been isolated, and those which have been reported are all highly divergent when compared to the retroviruses known to be harbored by mammals and birds. Here we show that retroviruses with significant homology to the human endogenous retrovirus type I (HERV-I) are present within the genomes of fish, reptiles, birds, and mammals and that they may well be widespread within many vertebrates. Phylogenetic analysis of nucleotide sequences strongly supported the inclusion of viruses from each of these vertebrate classes into one monophyletic group. This analysis also demonstrated that the HERV-I-related viruses are more closely related to retroviruses belonging to the murine leukemia virus genus than to members of the other retroviral genera. The presence of HERV-I-related retroviruses in so many disparate vertebrate hosts suggests that other endogenous human retroviruses may also have a much wider distribution than is currently appreciated.