bluuebunny/crossref_metadata_2025 huggingface dataset:
Replication-defective mutants of herpes simplex virus (HSV) induce cellular immunity and protect against lethal HSV infection
Authors: Nguyen L H , Knipe D M , Finberg R W
Published: January 2020

Live viruses and live virus vaccines induce cellular immunity more readily than do inactivated viruses or purified proteins, but the mechanism by which this process occurs is unknown. A trivial explanation would relate to the ability of live viruses to spread and infect more cells than can inactivated virus. We have used live but replication-defective mutants to investigate this question. Our studies indicate that the immune responses of mice to live virus differ greatly from the responses to inactivated virus even when the virus does not complete a replicative cycle. Further, these studies indicate that herpes simplex virus-specific T-cell responses can be generated by infection with replication-defective mutant viruses. These data indicate that the magnitude of the cellular immunity to herpes simplex virus may be proportional to the number or quantity of different viral gene products expressed by an immunizing virus.
The pseudorabies virus homology of the herpes simplex virus UL21 gene product is a capsid protein which is involved in capsid maturation
Authors: de Wind N , Wagenaar F , Pol J , Kimman T , Berns A
Published: January 2020

We mutagenized, mapped, and sequenced the pseudorabies virus (PRV) homology of gene UL21 of herpes simplex virus type 1. A polyclonal mouse antiserum against the protein encoded by the UL21 homolog was generated and used to monitor the expression and subcellular localization of the UL21-encoded protein. We found that the protein is identical to a previously detected PRV capsid protein. We analyzed viable PRV strains encoding mutant UL21 homologys, truncated by insertion of an oligonucleotide that contains stop codons in all reading frames. In two PRV mutants carrying the oligonucleotide at two sites within the gene, processing of newly replicated viral DNA was impaired. In addition, we show that one of the UL21 mutants has strongly reduced virulence for mice.
Multiple glycoproteins synthesized by the smallest RNA segment (S10) of bluetongue virus
Authors: Wu X , Chen S Y , Iwata H , Compans R W , Roy P
Published: January 2020

The genome of bluetongue virus, an orbivirus, consists of 10 double-stranded RNAs, each encoding at least one polypeptide. The smallest RNA segment (S10) encodes two minor nonstructural proteins, NS3 and NS3A, the structures and functions of which are not understood. We have expressed these two proteins in mammalian cells by using the T7 cytoplasmic transient expression system. Using a deletion mutant (lacking the first AUG initiation codon), we have demonstrated that the second initiation codon is used to initiate the synthesis of NS3A protein and that the two initiation codons are responsible for the synthesis not only of NS3 and NS3A but also of high-molecular-weight forms of both proteins. These higher-molecular-weight forms (GNS3 and GNS3A) are glycosylated. We have also demonstrated that the carbohydrate chains of GNS3 and GNS3A could be further modified by heterogeneous extension to polylactosaminoglycan forms. The glycosylated and nonglycosylated forms are found in similar intracellular locations in the Golgi complex. In the presence of cycloheximide, NS3 and NS3A immunofluorescence staining was pronounced in the Golgi complex, confirming that NS3 and NS3A are competent for transport to the Golgi apparatus after synthesis. We conclude that S10 gene products are integral membrane glycoproteins.
Baculovirus phosphoprotein pp31 is associated with virogenic stroma
Authors: Guarino L A , Dong W , Xu B , Broussard D R , Davis R W , Jarvis D L
Published: January 2020

The PstI K fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a protein with a molecular weight of 31,000. To define the role of this protein (pp31) in virus infection further, it was overexpressed in bacteria and used to produce polyclonal antiserum. Radioimmunoprecipitation analysis indicated that pp31 was synthesized during both the early and late phases of virus infection, consistent with previous analyses indicating that the gene was regulated by tandem early and late promoters. Metabolic labeling of cells with carrier-free phosphate indicated that pp31 was phosphorylated. Biochemical fractionation experiments showed that pp31 was localized in the nucleus and that it was more stably associated with the nucleus at later times of infection. Immunoblot analysis of subnuclear fractions indicated that pp31 was associated predominantly with the chromatin and nuclear matrix fractions. Immunofluorescence experiments confirmed that the pp31 protein was localized in the nucleus. Nuclear staining was relatively uniform early but was more centrally nuclear later in infection. Immunoelectron microscopy indicated that the pp31 protein was a component of virogenic stroma. Southwestern (DNA-protein) blot analysis demonstrated that pp31 is a DNA-binding protein. These findings suggest a possible role for pp31 in the virus life cycle.
Glycosylation requirements for intracellular transport and function of the hemagglutinin of influenza virus
Authors: Gallagher P J , Henneberry J M , Sambrook J F , Gething M J
Published: January 2020

The contribution of each of the seven asparagine-linked oligosaccharide side chains on the hemagglutinin of the A/Aichi/68 (X31) strain of influenza virus was assessed with respect to its effect on the folding, intracellular transport, and biological activities of the molecule. Twenty mutant influenza virus hemagglutinins were constructed and expressed, each of which had one or more of the seven glycosylation sites removed. Investigations of these mutant hemagglutinins indicated that (i) no individual oligosaccharide side chain is necessary or sufficient for the folding, intracellular transport, or function of the molecule, (ii) at least five oligosaccharide side chains are required for the X31 hemagglutinin molecule to move along the exocytic pathway to the plasma membrane, and (iii) mutant hemagglutinins having less than five oligosaccharide side chains form intracellular aggregates and are retained in the endoplasmic reticulum.
U3 long terminal repeat-mediated induction of intracellular immunity by a murine retrovirus: a novel model of latency for retroviruses
Authors: Gorska-Flipot I , Huang M , Cantin M , Rassart E , Massé G , Jolicoeur P
Published: January 2020

BL/VL3 radiation leukemia virus (RadLV) is a thymotropic, highly leukemogenic murine leukemia virus (MuLV) which is unable to replicate in vitro in mouse fibroblasts. We have previously reported that the U3 long terminal repeat region of its genome is responsible for this block (E. Rassart, Y. Paquette, and P. Jolicoeur, J. Virol. 62:3840-3848, 1988). By using hybrids of permissive and resistant cells infected with BL/VL3 RadLV or fibrotropic MuLV, we found that the resistant phenotype was dominant. Investigation to determine at which step of the virus cycle the block operates revealed that integration, transcription, and translation of the BL/VL3 viral genome occurred at normal levels in nonpermissive cells. The BL/VL3 RadLV Pr65gag proteins made in nonpermissive cells were also myristylated and located at the membrane, and the levels of their cleaved products were similar to those of fibrotropic MuLV. However, processing of BL/VL3 RadLV Pr85env was impaired in nonpermissive cells. Virions were not released into the culture medium of nonpermissive cells, as measured by reverse transcriptase activity and by content in p30 or gp70 protein and as documented by lower levels of budding particles seen by electron microscopy. These results indicate that BL/VL3 RadLV replication is blocked at a late stage of the virus cycle, i.e., at virion assembly. Interestingly, these BL/VL3 RadLV-infected nonpermissive fibroblasts were resistant to superinfection by fibrotropic Moloney MuLV, and this resistance also occurred at a late step of the Moloney virus cycle. Since this block is dominant, it appears that the U3 long terminal repeat region of the BL/VL3 viral genome has the ability to induce a cellular suppressor factor(s), thus bringing intracellular immunity against itself and against other ecotropic MuLVs.
Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope
Authors: Isaacs S N , Wolffe E J , Payne L G , Moss B
Published: January 2020

Using a reverse genetic approach, we have demonstrated that the product of the B5R open reading frame (ORF), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (EEV) form of vaccinia virus but absent from the intracellular naked (INV) form. An antibody (C'-B5R) raised to a 15-amino-acid peptide from the translated B5R ORF reacted with a 42-kDa protein (gp42) found in vaccinia virus-infected cells and cesium chloride-banded EEV but not INV. Under nonreducing conditions, an 85-kDa component, possibly representing a hetero- or homodimeric form of gp42, was detected by both immunoprecipitation and Western immunoblot analysis. Metabolic labeling with [3H]glucosamine and [3H]palmitate revealed that the B5R product is glycosylated and acylated. The C-terminal transmembrane domain of the protein was identified by constructing a recombinant vaccinia virus that overexpressed a truncated, secreted form of the B5R ORF product. By N-terminal sequence analysis of this secreted protein, the site of signal peptide cleavage of gp42 was determined. A previously described monoclonal antibody (MAb 20) raised to EEV, which immunoprecipitated a protein with biochemical characteristics similar to those of wild-type gp42, reacted with the recombinant, secreted product of the B5R ORF. Immunofluorescence of wild-type vaccinia virus-infected cells by using either MAb 20 or C'-B5R revealed that the protein is expressed on the cell surface and within the cytoplasm. Immunogold labeling of EEV and INV with MAb 20 demonstrated that the protein was found exclusively on the EEV membrane.
Mutational analysis of the octapeptide sequence motif at the NS1-NS2A cleavage junction of dengue type 4 virus
Authors: Pethel M , Falgout B , Lai C J
Published: January 2020

We have previously shown that proper processing of dengue type 4 virus NS1 from the NS1-NS2A region of the viral polyprotein requires a hydrophobic N-terminal signal and the downstream NS2A. Results from deletion analysis indicate that a minimum length of eight amino acids at the C terminus of NS1 is required for cleavage at the NS1-NS2A junction. Comparison of this eight-amino-acid sequence with the corresponding sequences of other flaviviruses suggests a consensus cleavage sequence of Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala. Site-directed mutagenesis was performed to construct mutants of NS1-NS2A that contained a single amino acid substitution at different positions of the consensus cleavage sequence or at the immediate downstream position. Three to eight different substitutions were made at each position. A total of 50 NS1-NS2A mutants were analyzed for their cleavage efficiency relative to that of the wild-type dengue type 4 virus sequence. As predicted, nearly all substitutions at positions P1, P3, P5, P7, and P8, occupied by conserved amino acids, yielded low levels of cleavage, with the exception that Pro or Ala substituting for Ser (P5) was tolerated. Substitutions of an amino acid at the remaining positions occupied by nonconserved amino acids generally yielded high levels of cleavage. However, some substitutions at nonconserved positions were not tolerated. For example, substitution of Gly or Glu for Gln (P4) and substitution of Val or Glu for Lys (P6) each yielded a low level of cleavage. Overall, these data support the proposed cleavage sequence motif deduced by comparison of sequences among the flaviviruses. This study also showed that in addition to the eight-amino-acid sequence, the amino acid immediately following the NS1-NS2A cleavage site plays a role in cleavage.
Characterization of reticuloendotheliosis virus-transformed avian T-lymphoblastoid cell lines infected with Marek's disease virus
Authors: Pratt W D , Morgan R W , Schat K A
Published: January 2020

The expression of Marek's disease virus (MDV) transcripts and protein products was investigated in reticuloendotheliosis virus-transformed avian T-lymphoblastoid cell line RECC-CU91, which was superinfected with MDV. The presence of MDV in the superinfected cell line, renamed RECC-CU210, was demonstrated by Southern hybridization with 32P-labeled BamHI-H and -B fragments of the BamHI MDV DNA library. Examination of RECC-CU210 for the expression of MDV-specific RNA transcripts encoded by the internal repeat long (IRL), internal repeat short (IRS), and unique short (US) regions of the MDV genome revealed two small transcripts of 0.6 and 0.7 kb. These transcripts were mapped to the IRL and IRS regions, respectively. In contrast, RECC-CU211, which was developed through transfection of CU210 with the BamHI-A fragment of MDV, expressed an additional nine transcripts from the IRL, IRS, and US regions. CU211 but not CU210 also expressed a complex of polypeptides of 40, 38, and 24 kDa, identified by monoclonal antibodies as MDV-specific phosphoproteins. The 38-kDa phosphoprotein is likely to be pp38, an early viral protein that maps within the IRL region of the MDV genome. These findings suggest that genes located within the transfected BamHI-A fragment transactivated a number of genes located in the IRL region of the MDV genome.
The Epstein-Barr virus immediate-early promoter BRLF1 can be activated by the cellular Sp1 transcription factor
Authors: Zalani S , Holley-Guthrie E A , Gutsch D E , Kenney S C
Published: January 2020

Disruption of viral latency in Epstein-Barr virus-infected cells is mediated through the activation of the BZLF1 (Z) immediate-early gene product. The Z protein can be derived from either of two promoters: the BZLF1 promoter, which directs transcription of a 1.0-kb mRNA encoding the Z gene product alone, or the upstream BRLF1 promoter, which directs transcription of a 2.8-kb bicistronic mRNA encoding the BRLF1 and BZLF1 immediate-early proteins. In this study we have examined the regulation of the BRLF1 promoter by viral and cellular factors. We found that the BRLF1 promoter is autoregulated by the BRLF1 transactivator through a nonbinding mechanism. We show that the BRLF1 (but not the BZLF1) promoter is highly responsive to the Sp1 transcription factor. Sp1 activation of the BRLF1 promoter is mediated through a consensus Sp1-binding site located from -39 to -44 (relative to the mRNA start site). We demonstrate that the BRLF1 promoter has high constitutive activity in C-33 cells (an epithelial cell line) and that the proximal Sp1-binding site is required for this activity. Despite the ubiquitous presence of Sp1 in many cell types, we found that the BRLF1 promoter has essentially no activity in lymphoid cell lines, suggesting that factors other than Sp1 may negatively regulate the BRLF1 promoter in these cells. Our findings demonstrate that the two potential promoters directing BZLF1 transcription are differentially regulated and that Sp1 can activate the BRLF1 promoter but not the BZLF1 promoter.
Isolation and characterization of cDNAs from BamHI-H gene family RNAs associated with the tumorigenicity of Marek's disease virus
Authors: Peng F , Bradley G , Tanaka A , Lancz G , Nonoyama M
Published: January 2020

It has been reported that loss of the tumorigenic potential of attenuated Marek's disease virus (MDV) is strongly associated with amplification of the 132-bp repeat sequences found within the BamHI-D and BamHI-H fragments contained within the long terminal repeat and the long internal repeat, respectively. The expansion of this region results in loss of transcripts that are 3.8, 3.0, and 1.8 kbp long that are produced by tumorigenic strains of MDV. This evidence suggests that production of one or more of these three RNAs is strongly associated with the tumorigenic potential of the virus. In this study, we have cloned and sequenced 1.69-, 1.5-, 1.9-, and 2.2-kbp cDNAs from the BamHI-H gene family RNAs associated with tumorigenicity. The 1.69- and 2.2-kbp cDNAs are derived from nonspliced transcripts, whereas the 1.5- and 1.9-kbp cDNAs are from single spliced mRNAs spanning the BamHI-H and BamHI-I2 fragments of MDV DNA. Sequence analysis has shown two potential open reading frames in each of the cDNAs. The putative 63-amino-acid protein encoded by the first open reading frame in the 1.69-kbp cDNA and a putative 75-amino-acid protein encoded by the first open reading frame in the 1.5-kbp cDNA showed limited homology with the mouse T-cell lymphoma oncogene and the fes/fps family of kinase-related transforming proteins.
Creation and characterization of temperature-sensitive mutants of the lck tyrosine protein kinase
Authors: Hurley T R , Amrein K E , Sefton B M
Published: January 2020

Temperature-sensitive mutants of the lck tyrosine protein kinase were created by the introduction of mutations known to cause temperature sensitivity of the v-src tyrosine protein kinase of Rous sarcoma virus. p56lck activated by mutation of the regulatory site of tyrosine phosphorylation, Tyr-505, to Phe transforms fibroblasts in culture. Mutations identical to those responsible for the temperature-sensitive phenotypes of the tsNY68 and tsNY72-4 v-src mutants rendered this activated lck gene temperature sensitive for both morphological transformation and induction of growth in soft agar. The mutant proteins were incapable of cellular transformation at the nonpermissive temperature in part because of failure of the lck protein to accumulate to normal levels. Morphological transformation of fibroblasts was detectable within 24 h of a shift of cells to the permissive temperature and was essentially complete in 48 to 72 h. These mutants should prove useful for the study of the function of the lck kinase in hematopoietic cells.
Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells
Authors: LaFemina R L , Schneider C L , Robbins H L , Callahan P L , LeGrow K , Roth E , Schleif W A , Emini E A
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) integrase enzyme exhibits significant amino acid sequence conservation with integrase proteins of other retroviruses. We introduced specific amino acid substitutions at a number of the conserved residue positions of recombinant HIV-1 integrase. Some of these substitutions resulted in proteins which were not able to be purified in the same manner as the wild-type enzyme, and these were not studied further. The remaining mutant enzymes were assessed for their abilities to perform functions characteristic of the integrase protein. These included specific removal of the terminal dinucleotides from oligonucleotide substrates representative of the viral U5-long terminal repeat, nonspecific cleavage of oligonucleotide substrates, and mediation of the strand transfer (integration) reaction. Substitution at position 43, within the protein's zinc finger motif region, resulted in an enzyme with reduced specificity for cleavage of the terminal dinucleotide. In addition, a double substitution of aspartic acid and glutamine for valine and glutamic acid, respectively, at positions 151 and 152 within the D,D(35)E motif region rendered the integrase protein inactive for all of its functions. The introduction of this double substitution into an infectious HIV-1 provirus yielded a mutant virus that was incapable of productively infecting human T-lymphoid cells in culture.
Coronavirus species specificity: murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein
Authors: Compton S R , Stephensen C B , Snyder S W , Weismiller D G , Holmes K V
Published: January 2020

Like most coronaviruses, the coronavirus mouse hepatitis virus (MHV) exhibits strong species specificity, causing natural infection only in mice. MHV-A59 virions use as a receptor a 110- to 120-kDa glycoprotein (MHVR) in the carcinoembryonic antigen (CEA) family of glycoproteins (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G. S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; and R. K. Williams, G. S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). The role of virus-receptor interactions in determining the species specificity of MHV-A59 was examined by comparing the binding of virus and antireceptor antibodies to cell lines and intestinal brush border membranes (BBM) from many species. Polyclonal antireceptor antiserum (anti-MHVR) raised by immunization of SJL/J mice with BALB/c BBM recognized MHVR specifically in immunoblots of BALB/c BBM but not in BBM from adult SJL/J mice that are resistant to infection with MHV-A59, indicating a major difference in epitopes between MHVR and its SJL/J homolog which does not bind MHV (7). Anti-MHVR bound to plasma membranes of MHV-susceptible murine cell lines but not to membranes of human, cat, dog, monkey, or hamster cell lines. Cell lines from these species were resistant to MHV-A59 infection, and only the murine cell lines tested were susceptible. Pretreatment of murine fibroblasts with anti-MHVR prevented binding of radiolabeled virions to murine cells and prevented virus infection. Solid-phase virus-binding assays and virus overlay protein blot assays showed that MHV-A59 virions bound to MHVR on intestinal BBM from MHV-susceptible mouse strains but not to proteins on intestinal BBM from humans, cats, dogs, pigs, cows, rabbits, rats, cotton rats, or chickens. In immunoblots of BBM from these species, both polyclonal and monoclonal antireceptor antibodies that block MHV-A59 infection of murine cells recognized only the murine CEA-related glycoprotein and not homologous CEA-related glycoproteins of other species. These results suggest that MHV-A59 binds to a mouse-specific epitope of MHVR, and they support the hypothesis that the species specificity of MHV-A59 infection may be due to the specificity of the virus-receptor interaction.
Loss of infectivity by progeny virus from alpha interferon-treated human immunodeficiency virus type 1-infected T cells is associated with defective assembly of envelope gp120
Authors: Hansen B D , Nara P L , Maheshwari R K , Sidhu G S , Bernbaum J G , Hoekzema D , Meltzer M S , Gendelman H E
Published: January 2020

Levels of human immunodeficiency virus (HIV) DNA, RNA, or p24 antigen and reverse transcriptase activity in T-cell cultures treated with 500 IU of recombinant alpha interferon (rIFN alpha) per ml were comparable to those in control cultures. Radioimmunoprecipitation analysis of proteins in lysates of IFN-treated T cells documented a marked accumulation of HIV proteins. Localization of gp120 by immunofluorescence showed a diffuse pattern in IFN-treated cells quite distinct from the ring pattern in untreated control cells. That large quantities of gp120 in aberrant cell compartments might affect HIV morphogenesis was confirmed in infectivity studies: virions from IFN-treated cells were 100- to 1,000-fold less infectious than an equal number of virions from control cells. Direct examination of IFN-treated and control HIV-infected cells by transmission electron microscopy showed little difference in the number or distribution of viral particles. However, quantitation of gp120 by immunogold particle analysis revealed a marked depletion of envelope glycoprotein in virions released from IFN-treated cells. This defect in gp120 assembly onto mature viral particles provides a molecular basis for this loss of infectivity.
Mutagenesis of the Glu-89 residue in human immunodeficiency virus type 1 (HIV-1) and HIV-2 reverse transcriptases: effects on nucleoside analog resistance
Authors: Song Q , Yang G , Goff S P , Prasad V R
Published: January 2020

A Glu-89-->Gly alteration in the human immunodeficiency virus type 1 reverse transcriptase (RT) was previously shown to result in resistance to several dideoxynucleoside analogs and to phosphonoformic acid (PFA; foscarnet). This residue was altered to Ala, Val, Ser, Thr, Gln, Asp, Asn, or Lys, and the ddGTP and PFA sensitivities of the mutant RTs were measured. Replacements with Ala, Gly, Val, and Thr led to resistance to inhibition by ddGTP, while mutants with amino acid Ser, Gln, Asn, Asp, or Lys displayed only moderate or no resistance. A similar result was obtained with inhibition by PFA, except that the Asp-89 mutant also displayed resistance. Furthermore, the introduction of Glu-89-->Gly alteration into the RT of human immunodeficiency virus type 2 likewise rendered it resistant to both ddGTP and PFA.
Upstream sequences and cap proximity in the regulation of polyadenylation in ground squirrel hepatitis virus
Authors: Cherrington J , Russnak R , Ganem D
Published: January 2020

The polyadenylation signal of mammalian hepadnaviruses is unusual in that its hexanucleotide element is the variant UAUAAA rather than AAUAAA. This signal functions inefficiently and must be augmented by multiple activator elements located in the upstream 400 nucleotides (nt) to promote efficient processing. Here we characterize one of these upstream elements, termed PS2, in the ground squirrel hepatitis virus. PS2 is located within the 107 nt 5' to the UAUAAA and raises the efficiency of polyadenylation by this signal from < 10% to 50 to 60%. It can function independently of the more 5' activator elements and conversely is not required for their function. Its action is orientation dependent, and a predicted stem-loop structure within the element is not necessary for its activity. PS2 is the sole upstream element that maps within the terminal redundancy of viral genomic RNA. Thus, it is present, together with the UAUAAA, at both the 5' and 3' ends of this RNA. During genomic RNA synthesis, the poly(A) signals in the 5' repeat are bypassed, while those in the 3' copy are used. The ability of PS2 to function independently of the other, more upstream activators suggests that the absence of the latter elements from the 5' redundancy is insufficient to account for bypass of the 5' poly(A) site, as we had earlier proposed. Rather, the short distance from the cap site to the UAUAAA at the 5' end of genomic RNA actively suppresses its use, as this suppression can be experimentally relieved by increasing this distance to 230 to 400 nt.
Cassette mutagenesis of the reverse transcriptase of human immunodeficiency virus type 1
Authors: Boyer P L , Ferris A L , Hughes S H
Published: January 2020

We constructed a series of BspMI cassettes that simplify the introduction of specific point mutations in the polymerase domain of human immunodeficiency virus type 1 reverse transcriptase. A series of point mutants were constructed by using these cassette vectors. The RNA-dependent DNA polymerase and RNase H activities of 20 point mutations in the conserved portion of the polymerase domain were assayed. All the mutations analyzed are conservative substitutions of evolutionarily conserved amino acids. The mutations were divided into four classes. The first class has little effect on either polymerase or RNase H activity. The second class affects RNase H but not polymerase activity, while the third class has a normal RNase H activity with diminished polymerase activity. The fourth class affects both activities.
Transactivation of the cytomegalovirus ICP36 gene promoter requires the alpha gene product TRS1 in addition to IE1 and IE2
Authors: Stasiak P C , Mocarski E S
Published: January 2020

Very little is known about the human cytomegalovirus functions that activate gamma (late) gene expression. We have investigated the regulation of the human cytomegalovirus gamma gene encoding the ICP36 major late DNA-binding protein family (UL44). Transactivation of the ICP36 gene promoter was found to be absolutely dependent on the trs1 gene product when expressed in cells in conjunction with ie1 and ie2 gene products. Transactivation occurred poorly or not at all when any one of these three transactivators was omitted. TRS1 is a member of the US22 family of proteins and is encoded by a region near the L-S junction of the viral genome within the c repeat and adjacent Us sequences. TRS1 is highly homologous to IRS1, which is encoded from the other copy of the c repeat, and plasmid constructs carrying the irs1 gene were also able to mediate transactivation of the ICP36 promoter. RNA blot analysis of steady-rate RNA throughout infection showed that the trs1 transcript was expressed with the kinetics of an alpha gene but its accumulation was delayed relative to that of ie1 and ie2 transcripts. On the basis of these experiments, TRS1 and IRS1 are proposed to be important intermediaries in the cascade of cytomegalovirus gene expression.
Simian virus 40 large T-antigen expression decreases the G1 and increases the G2 + M cell cycle phase durations in exponentially growing cells
Authors: Sladek T L , Jacobberger J W
Published: January 2020

The effect of simian virus 40 large T-antigen (Tag) expression on the cell cycle of exponentially growing, established, mouse NIH 3T3 fibroblasts was examined by using a sensitive flow cytometric assay to analyze nonselected cells immediately after infection with a Tag-encoding recombinant retrovirus. Tag expression resulted in reduced percentages of G1-phase cells and increased percentages of S- and G2 + M-phase cells compared with cell populations infected with a control virus not encoding the Tag gene. Cell cycle-blocking drugs were used to examine the exit rate for each of the cell cycle phases, G1, S, and G2 + M, for Tag-expressing and Tag-nonexpressing cells growing in the same cell culture dish. As a result of Tag expression, the duration of the G1 phase was decreased (average G1-phase exit duration decreased by 18%) and the duration of the G2 + M phase was increased (average G2 + M exit duration increased by 29%). The duration of S phase was unaffected by Tag expression.
Hepatitis B virus (HBV)-specific cytotoxic T-cell (CTL) response in humans: characterization of HLA class II-restricted CTLs that recognize endogenously synthesized HBV envelope antigens
Authors: Penna A , Fowler P , Bertoletti A , Guilhot S , Moss B , Margolskee R F , Cavalli A , Valli A , Fiaccadori F , Chisari F V
Published: January 2020

In this study, we show that CD4+, hepatitis B virus (HBV) envelope-specific T-cell clones produced by stimulation with a particulate antigen preparation are able to recognize and kill not only autologous antigen-presenting cells incubated with exogenous HBV envelope antigens but also autologous HLA class II-positive cells expressing endogenously synthesized HBV envelope antigens following infection with recombinant vaccinia viruses or transfection with recombinant Epstein-Barr virus expression vectors. Experiments with lysosomotropic agents and brefeldin A suggest that the endosomal compartment is likely involved in the processing of endogenously synthesized viral proteins for recognition by CD4+ T cells. Our study indicates that HBV envelope-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes can potentially participate in the immune clearance of HBV-infected cells and the pathogenesis of hepatocellular injury in hepatitis B.
Human immunodeficiency virus type 1 and type 2 protease monomers are functionally interchangeable in the dimeric enzymes
Authors: Patterson C E , Seetharam R , Kettner C A , Cheng Y S
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 proteases are dimers of identical subunits. We made a construct for the expression of recombinant one-chain HIV-2 protease dimer, which, like the previously described one-chain HIV-1 protease dimer, is fully active. The constructs for the one-chain dimers of HIV-1 and HIV-2 proteases were modified to produce hybrid one-chain dimers consisting of both HIV-1 and HIV-2 protease monomers. Although the monomers share only 47.5% sequence identity, the hybrid one-chain dimers are fully active, suggesting that the folding of both HIV-1 and HIV-2 protease monomers is functionally similar.
Novel GACG-hairpin pair motif in the 5' untranslated region of type C retroviruses related to murine leukemia virus
Authors: Konings D A , Nash M A , Maizel J V , Arlinghaus R B
Published: January 2020

We searched for the presence of common RNA structural motifs in mammalian type C retroviruses related to murine leukemia viruses and the closely related avian spleen necrosis virus. A novel motif consisting of a pair of hairpins, called hairpin pair motif, was detected in the 5' untranslated regions of the genomes of these retroviruses. A combination of computational analyses that included the assessment of phylogenetic sequence conservation by multiple alignment, the search for regions with unusual RNA folding properties, and the analysis of RNA secondary structure by suboptimal free-energy calculations highlighted the significance of this hairpin pair motif. The hairpin pair motif encompasses 70 to 80 nucleotides between the splice donor site and the gag translational initiation codon of these viruses. The motif is composed of two adjacent hairpins both with a perfectly conserved GACG tetraloop. We propose that the novel GACG-hairpin pair motif described here constitutes an essential component of the regulatory machinery in these type C retroviruses.
Influence of adeno-associated virus on adherence and growth properties of normal cells
Authors: Bantel-Schaal U , Stöhr M
Published: January 2020

It has been shown previously that infection of newly established cell cultures from malignant human tumors with adeno-associated parvovirus type 2 or type 5 results in growth arrest and cell death. Here we report that the additionally observed antiproliferative effect on diploid human fibroblasts is transient and is connected to a reduced number of cells in S phase. Progression through the cell cycle is disturbed either in G0/G1 or at the G1/S boundary, but an additional arrest in G2 cannot be excluded. DNA synthesis and cell proliferation are resumed when cells are recultured after loosening of cell-matrix adhesions by trypsin treatment. In contrast, they are not resumed by solely providing growth factors via higher amounts of fetal calf serum. The results suggest that cell adherence is altered in adeno-associated parvovirus-infected human embryo fibroblasts.
Herpes simplex virus infection can occur without involvement of the fibroblast growth factor receptor
Authors: Muggeridge M I , Cohen G H , Eisenberg R J
Published: January 2020

Basic fibroblast growth factor (bFGF) has been reported to block uptake of herpes simplex virus type 1 (HSV-1) and plaque formation on arterial smooth muscle cells, suggesting a role for the bFGF receptor in HSV entry (R. J. Kaner, A. Baird, A. Mansukhani, C. Basilico, B. D. Summers, R. Z. Florkiewicz, and D. P. Hajjar, Science 248:1410-1413, 1990). We confirmed the effect of bFGF on infection of this cell type with HSV-1 and HSV-2 and found the same result with umbilical vein endothelial cells. However, bFGF does not inhibit plaque formation on any other cell type we tested. Furthermore, there is no correlation between the level of expression of the bFGF receptor and the effect of bFGF. HEp-2 and A431 cells express barely detectable levels of receptor, and yet they are fully permissive for HSV infection in a bFGF-insensitive manner. Thus, interaction of virus with the bFGF receptor is not required for infection of many cell types. In addition, infection of smooth muscle cells is not prevented by incubation of virus with an anti-bFGF antibody, arguing against the hypothesis that virion-associated bFGF acts as a bridge between virus and receptor (A. Baird, R. Z. Florkiewicz, P. A. Maher, R. J. Kaner, and D. P. Hajjar, Nature [London] 348:344-346, 1990).
Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein define a requirement for dibasic residues for intracellular cleavage
Authors: Dong J Y , Dubay J W , Perez L G , Hunter E
Published: January 2020

We investigated the amino acid sequence requirements for intracellular cleavage of the Rous sarcoma virus glycoprotein precursor by introducing mutations into the region encoding the cleavage recognition site (Arg-Arg-Lys-Arg). In addition to mutants G1 (Arg-Arg-Glu-Arg) and Dr1 (deletion of all four codons) that we have reported on previously (L. G. Perez and E. Hunter, J. Virol. 61:1609-1614, 1987), we constructed two additional mutants, AR1 (Arg-Arg-Arg-Arg), in which the highly conserved lysine is replaced by an arginine, and S19 (Ser-Arg-Glu-Arg), in which no dibasic pairs remain. The results of these studies demonstrate that when the cleavage sequence is deleted (Dr1) or modified to contain unpaired basic residues (S19), intracellular cleavage of the glycoprotein precursor is completely blocked. This demonstrates that the cellular endopeptidase responsible for cleavage has a stringent requirement for the presence of a pair of basic residues (Arg-Arg or Lys-Arg). Furthermore, it implies that the cleavage enzyme is not trypsinlike, since it is unable to recognize arginine residues that are sensitive to trypsin action. Substitution of the mutated genes into a replication-competent avian retrovirus genome showed that cleavage of the glycoprotein precursor was not required for incorporation into virions but was necessary for infectivity. Treatment of BH-RCAN-S19-transfected turkey cells with low levels of trypsin resulted in the release of infectious virus, demonstrating that exogenous cleavage could generate a biologically active glycoprotein molecule.
The third subunit of protein phosphatase 2A (PP2A), a 55-kilodalton protein which is apparently substituted for by T antigens in complexes with the 36- and 63-kilodalton PP2A subunits, bears little resemblance to T antigens
Authors: Pallas D C , Weller W , Jaspers S , Miller T B , Lane W S , Roberts T M
Published: January 2020

The small and middle T (tumor) antigens of polyomavirus have been shown previously to associate with the 36-kDa catalytic subunit and the 63-kDa regulatory subunit of protein phosphatase type 2A, apparently substituting for a normal third 55-kDa regulatory subunit (D.C. Pallas, L.K. Shahrik, B.L. Martin, S. Jaspers, T.B. Miller, D.L. Brautigan, and T.M. Roberts, Cell 60:167-176, 1990). To facilitate a comparison of the normal regulatory subunit and T antigens, we isolated a 2.14-kb cDNA clone encoding this 55-kDa subunit from a rat liver library. Using a probe from the coding region of this gene, we detected a major 2.4-kb mRNA transcript in liver and muscle RNAs. The 55-kDa protein phosphatase 2A subunit purified from rat skeletal muscle generates multiple species when analyzed on two-dimensional gels. Transcription and translation of the clone in vitro produced a full-length protein that comigrated precisely on two-dimensional gels with three of these species, indicating that the 55-kDa protein is apparently modified similarly in vivo and in reticulocyte lysates. Additional species in the purified preparation were not found in the translate, suggesting that there are probably two or more isoforms of this protein in rat muscle. Somewhat surprisingly, there was no clear homology with T-antigen amino acid sequences.
Characterization of the regulatory functions of the equine herpesvirus 1 immediate-early gene product
Authors: Smith R H , Caughman G B , O'Callaghan D J
Published: January 2020

Use of the translation-inhibiting drug cycloheximide has indicated that the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene, the sole EHV-1 IE gene, encodes a major viral regulatory protein since IE mRNA translation is a prerequisite for all further viral gene expression (W.L. Gray, R. P. Baumann, A. T. Robertson, G. B. Caughman, D. J. O'Callaghan, and J. Staczek, Virology 158:79-87, 1987). An EHV-1 IE gene expression vector (pSVIE) in combination with chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs was used in transient transfection assays to characterize the regulatory functions of the IE gene product. These experiments demonstrated that (i) the EHV-1 IE gene product is a bifunctional protein capable of both positive and negative modulation of gene expression; (ii) the IE gene product possesses an autoregulatory function which represses the IE promoter; (iii) IE autoregulation is dependent on IE promoter sequences mapping within positions -288 to +73 relative to the transcription initiation site (+1) of the IE gene; (iv) the IE gene product can independently activate the EHV-1 tk promoter (an early promoter) by as much as 60-fold; (v) two EHV-1 beta-gamma (leaky late) promoters, those of IR5 (gene 5 in the inverted repeat) and the glycoprotein D gene, demonstrate a requirement for both the IE gene product as well as a gene product encoded within the EHV-1 XbaI G fragment for significant activation; and (vi) the IE gene product is capable of activating heterologous viral promoters.
gag as well as myc sequences contribute to the transforming phenotype of the avian retrovirus FH3
Authors: Tikhonenko A T , Linial M L
Published: January 2020

The avian retrovirus FH3, like MC29 and CMII, encodes a Gag-Myc fusion protein. However, the FH3-encoded protein is larger, about 145 kDa, and contains almost the entire retroviral gag gene. In contrast to the other gag-myc avian retroviruses, FH3 fails to transform fibroblasts in vitro, although macrophages are transformed both in vitro and in vivo (C. Chen, B. J. Biegalke, R. N. Eisenman, and M. L. Linial, J. Virol. 63:5092-5100, 1989). We have used the polymerase chain reaction technique to obtain a molecular clone of FH3. Sequence analysis of the FH3 myc oncogene revealed a single proline----histidine change (position 223) relative to c-myc. However, substitution of the FH3 myc sequence with the chicken c-myc sequence did not alter the transformation potential of the virus. Hence, overexpression of the proto-oncogene as a Gag-Myc retroviral protein is sufficient for macrophage, but not fibroblast, transformation. After passage of FH3 in fibroblast cultures, a virus (FH3L) that is capable of rapidly transforming fibroblasts appears. The Gag-Myc protein encoded by FH3L is smaller (ca. 130 kDa) than that encoded by the original viral stock (FH3E). Sequencing of an FH3L molecular clone revealed a 212-amino-acid deletion within the Gag portion. Using FH3E/FH3L recombinants, we have demonstrated that the ability of encoded viruses to transform fibroblasts directly correlates with the presence of this deletion. Moreover, the addition of the Gag sequence deleted from FH3L to the MC29 oncoprotein significantly reduces its transforming activity as measured by focus assay. These data suggest that the C-terminal segment of Gag attenuates the oncogenic potential of Gag-Myc fusion proteins.
Identification and expression of rpo19, a vaccinia virus gene encoding a 19-kilodalton DNA-dependent RNA polymerase subunit
Authors: Ahn B Y , Rosel J , Cole N B , Moss B
Published: January 2020

The vaccinia virus DNA-dependent RNA polymerase subunit gene rpo19 was identified, and its expression was examined at RNA and protein levels. Antibody to the multisubunit RNA polymerase purified from virions reacted with a polypeptide with an apparent Mr of 21,000 that was synthesized in reticulocyte lysates programmed with (i) mRNA from infected cells that was isolated by hybridization to DNA subclones of the viral genomic HindIII A fragment and (ii) mRNA made in vitro by transcription of the viral open reading frame A6R. Polyclonal antiserum, raised to a recombinant protein product of the A6R open reading frame which could encode an 18,996-Da protein with an acidic N terminus, reacted with Mr-21,000 and -22,000 polypeptides that cosedimented with purified RNA polymerase. Internal sequencing of the two polypeptides confirmed that both were encoded by A6R, and the gene was named rpo19 to indicate the predicted molecular mass of the polypeptide in kilodaltons. Immunoblotting and metabolic labeling of infected cell proteins indicated that synthesis of the Mr-21,000 polypeptide started early and continued throughout virus infection, whereas the Mr-22,000 form appeared late following DNA replication. RNA analyses suggested that the rpo19 mRNA was expressed from a dual early/late promoter and that the protein-coding region of the mRNA was directly preceded by a short 5' poly(A) leader, apparently initiated within the TAAATG motif at the beginning of the open reading frame.
The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific
Authors: Urban M , Britt W , Mach M
Published: January 2020

Bacterial fusion proteins, constructed from overlapping fragments of the open reading frame coding for gp86 of human cytomegalovirus (HCMV) strain AD169, were used to localize antigenic regions recognized by antibodies from human convalescent sera. A major domain for binding of conformation-independent antibodies was localized on fusion protein AP86, containing amino acids 15 to 142 of gp86. Human antibodies, affinity purified on AP86, neutralized infectious virus in tissue culture. In addition, a mouse monoclonal antibody (AP86-SA4), raised against AP86, also neutralized HCMV. AP86-SA4 was reactive with viral gp86 in immunoblot assays and showed a plasma membrane staining on intact HCMV-infected fibroblasts late in infection. After exonuclease III deletions of the viral gene, the binding site of neutralizing human as well as mouse antibodies was localized between amino acid residues 34 and 43. The domain has sequence variation between laboratory strains AD169 and Towne, and binding of the antibodies was strain specific. To our knowledge, this is the first characterization of a strain-specific neutralizing epitope on HCMV.
Alpha interferon inhibits early stages of the human immunodeficiency virus type 1 replication cycle
Authors: Shirazi Y , Pitha P M
Published: January 2020

In this study, we have analyzed the effect of human alpha interferon (IFN-alpha) on a single replication cycle of human immunodeficiency virus type 1 (HIV-1) infection in the lymphocytic cell line CEM-174, which is highly sensitive to the antiviral effects of IFN. Pretreatment of cells with 50 to 500 U of recombinant human IFN-alpha per ml resulted in a marked reduction in viral RNA and protein synthesis. The effect of IFN-alpha was dose dependent and was amplified in multiple infection cycles. IFN-induced inhibition of viral protein synthesis could be detected only when cells were treated with IFN-alpha prior to infection or when IFN-alpha was added up to 10 h postinfection, but not if IFN-alpha was added at the later stages of HIV-1 replication cycle or after the HIV-1 infection was already established. Analysis of the integrated HIV-1 provirus showed a marked decrease in the levels of proviral DNA in IFN-treated cells. Thus, in contrast to the previous studies on established HIV-1 infection in T cells, in which the IFN block appeared to be at the posttranslational level, during de novo infection, IFN-alpha interferes with an early step of HIV-1 replication cycle that occurs prior to the integration of the proviral DNA. These results indicate that the early IFN block of HIV-1 replication, which has been previously observed only in primary marcophages, can also be detected in the IFN-sensitive T cells, indicating that the early IFN block is not limited to macrophages.