bluuebunny/crossref_metadata_2025 huggingface dataset:
The inability of human immunodeficiency virus to infect chimpanzee monocytes can be overcome by serial viral passage in vivo
Authors: Gendelman H E , Ehrlich G D , Baca L M , Conley S , Ribas J , Kalter D C , Meltzer M S , Poiesz B J , Nara P
Published: January 2020

Studies of lentivirus infection in ruminants, nonhuman primates, and humans suggest that virus infection of macrophages plays a central role in the disease process. To investigate whether human immunodeficiency virus type 1 (HIV-1) can infect chimpanzee macrophages, we recovered monocytes from peripheral blood mononuclear cells of HIV-1-negative animals and inoculated these and control human monocytes with a panel of four human-passaged monocytotropic virus strains and one chimpanzee-passaged isolate. HIV-1 infected human monocytes synthesized proviral DNA, viral mRNA, p24 antigen, and progeny virions. In contrast, except for the chimpanzee-passaged HIV-1 isolate, chimpanzee monocytes failed to support HIV-1 replication when cultured under both identical and a variety of other conditions. Proviral DNA was demonstrated only at background levels in these cell cultures by polymerase chain reaction for gag- and env-related sequences. Interestingly, the chimpanzee-passaged HIV-1 isolate did not replicate in human monocytes; viral p24 antigens and progeny virions were not detected. The same monocytotropic panel of HIV-1 strains replicated in both human and chimpanzee CD4+ T lymphoblasts treated with phytohemagglutinin and interleukin-2. The failure of HIV-1 to infect chimpanzee monocytes, which can be overcome by serial in vivo viral passage, occurs through a block early in the viral life cycle.
Identification of a strong enhancer element upstream from the pregenomic RNA start site of the duck hepatitis B virus genome
Authors: Crescenzo-Chaigne B , Pillot J , Lilienbaum A , Levrero M , Elfassi E
Published: January 2020

The genome of the duck hepatitis B virus (DHBV) contains an enhancer element. This sequence, of 192 bp, is located in the 3'-terminal coding region of the DNA polymerase gene (nucleotides 2159 to 2351), upstream from the pregenomic RNA start site. This enhancer potentiates a marked increased activity from the heterologous thymidine kinase promoter in an orientation-independent manner and at a proximal, as well as a distal, location. The DHBV enhancer activates transcription in a relatively cell-type-independent manner. Sequence homologies with the nuclear factor EF-C binding site are located in the DHBV enhancer. By using the HepG2 nuclear extracts and the DHBV enhancer as probes, a complex was observed in mobility shift assays.
Myristylation is involved in intracellular retention of hepatitis B virus envelope proteins
Authors: Prange R , Clemen A , Streeck R E
Published: January 2020

The envelope of hepatitis B virus contains three related proteins, one of which is myristylated. The nonmyristylated small and middle protein are assembled into empty envelope particles which are secreted from cells, whereas the myristylated large envelope protein is mainly found in complete virions and is not secreted in the absence of the nucleocapsid. The block to secretion can be partially overcome by mutation or deletion of the myristylation site. Creation of a myristyl attachment site in the small protein impairs the secretion of empty envelope particles but not their intracellular assembly. Myristylation may therefore play a crucial role in hepatitis B virus replication by channeling the envelope proteins into complete viral particles.
v-myb and v-ets cooperate for the mitogenic stimulation of primary fibroblasts by avian E26 retrovirus
Authors: Ravel-Chapuis P , Leprince D , Pain B , Li R , Domenget C , Stehelin D , Samarut J , Jurdic P
Published: January 2020

By using a series of deletion mutants, we have shown that the stimulation of fibroblast growth by E26 requires the cooperation of the two oncogenes, v-myb and v-ets, fused in the nuclear viral product. Of the two DNA-binding domains, only one must be present to promote anchorage-independent growth, whereas that of v-myb is required to allow growth in low serum medium. Furthermore, the v-ets oncogene comprises multifunctional domains.
Divergent molecular pathways of productive and latent infection with a virulent strain of herpes simplex virus type 1
Authors: Speck P G , Simmons A
Published: January 2020

Mutants of herpes simplex virus (HSV) have been used to show that a variety of key genes associated with initiation of lytic infection or replication of viral DNA are not essential for establishment of latency. These observations are extended in the present study, in which a virulent strain of HSV type 1 that is not compromised in its ability to productively infect neurons under favorable conditions was used to demonstrate early divergence of molecular pathways leading to productive and latent infection. Our experimental strategy made unique use of the segmental innervation of the vertebrate trunk to study the spread of virus throughout the peripheral nervous system after inoculation of mouse flanks. Evidence of viral gene expression, including that of immediate-early genes, was transient, confined to ganglia directly innervating the inoculated skin (8th through 12th thoracic segments), and seen only at sites from which infectious virus could be recovered. In contrast, neurons containing latency-associated transcripts and reactivatable virus were more widely distributed (sixth thoracic through first lumbar segments), from which we conclude that replication-competent HSV type 1 can establish latency without initiating productive infection.
Identification of upstream promoter elements mediating early transcription from the 35,000-molecular-weight protein gene of Autographa californica nuclear polyhedrosis virus
Authors: Dickson J A , Friesen P D
Published: January 2020

Site-directed mutagenesis was used to examine the organization of cis-acting regulatory elements that comprise the promoter of the early 35,000-molecular-weight protein gene (35K protein gene) encoded by the EcoRI-S region of the baculovirus Autographa californica nuclear polyhedrosis virus. The promoter fragment, extending from positions -226 to +12 relative to the early RNA start site (position +1), was fused to the reporter gene encoding chloramphenicol acetyltransferase (CAT) and then inserted into the genome of recombinant viruses (3.96 map units) in order to ascertain the role of regulatory elements in the context of a normal infection. A combination of deletions and linker insertions revealed that early transcription was mediated by a basal (minimum) promoter, consisting of the TATA element (positions -30 to -25), that was in turn responsive to an upstream activating region located between -90 and -30. The TATA element exerted the single greatest influence on the level of early promoter activity and contained all information necessary to direct transcription from a site located 30 nucleotides downstream. The upstream activating region provided a 10- to 15-fold stimulation of transcription from the early +1 start site that was mediated by distinct DNA elements. These regulatory elements included two GC motifs (centered at positions -81 and -54, respectively), composed of alternating G and C residues, and a CGT motif (position -40) that contained the core sequence A(A/T)CGT(G/T). Each motif was required for full promoter activity during the early phase of infection. This organization that employs diverse cis-acting stimulatory elements is typical of promoters responsive to RNA polymerase II and may facilitate increased expression of A. californica nuclear polyhedrosis virus genes early in infection when the level of viral DNA for transcription is critically low.
Biologic properties and nucleotide sequence analysis of human papillomavirus type 51
Authors: Lungu O , Crum C P , Silverstein S
Published: January 2020

Human papillomaviruses (HPVs) may be grouped according to the site from which they are isolated and the disease with which they are associated. We recently identified and cloned HPV type 51 (HPV-51) from a low-grade precancerous lesion (G. Nuovo, E. DeVilliers, R. Levine, S. Silverstein, and C. Crum. J. Virol. 62:1452-1455, 1988). Molecular epidemiologic analysis of cervical lesions, including condylomata and low- and high-grade precancers, revealed that HPV-51 was present in about 5% of the samples we examined. We have now determined the complete nucleotide sequence of this virus and compared it with other sequenced HPVs. Our analysis reveals that the 7,808-bp genome is composed of eight open reading frames which are encoded on the same strand and that this virus is most closely related to HPV-31. Sequence comparisons place this virus in the group of high-risk viruses (those with an increased risk of progressing to malignancy) along with HPV-16, -18, -31, and -33. Morphologic transformation experiments demonstrated that HPV-51 had transformation potential and that transformed cells contained RNAs homologous to E6 and E7.
Parvovirus NS1 stimulates P4 expression by interaction with the terminal repeats and through DNA amplification
Authors: Hanson N D , Rhode S L
Published: January 2020

Parvovirus protein NS1 is required for replication of viral DNA and plays a role in the regulation of viral gene expression. NS1 trans-activates the P38 promoter for capsid protein synthesis and has variable effects on other promoters. In this study, we examined the effects of NS1 on the regulation of its own promoter, P4. A number of plasmid constructions were made with the P4 promoter fused to reporter genes. The effects of NS1 on expression from the P4 promoter differed depending on the construction. Plasmids containing viral sequences which could not replicate showed a decrease in P4 expression on cotransfection with the NS1 gene. However, plasmids having replication-proficient viral sequences showed a three- to fivefold increase in P4 expression dependent on NS1. The effect on NS1 on P4 transcription was also evaluated at the steady-state RNA level. An infectious clone of the LuIII viral genome was modified to an NS1-NS2 null mutant (pLu272) that is competent for viral DNA replication by introducing a frameshift mutation at codon 5 of the NS1 open reading frame. The P4 transcripts of pLu272 are four nucleotides longer than the wild type and can therefore be resolved from the wild type by primer extension analysis. pLu272 allows comparison of the constitutive level of steady-state RNA produced by the pLu272 P4 promoter in the absence or presence of a template replication dependent on NS1 supplied in trans. NS1 increased P4 transcripts about six- to eightfold. Expression of P4 transcripts from clones that could not amplify depended on the presence of an intact inverted terminal repeat sequence at the left end. A clone with an intact viral left end and a defective viral right end gave an NS1-dependent threefold increase in P4 expression. Destruction of terminal hairpins at both ends resulted in no significant increase in P4 expression in the presence of NS1. Thus, the positive effect of NS1 on the steady-state levels of P4 transcripts depends on the amplification of gene copy number and the integrity of the terminal repeats.
Bone marrow depletion by 89Sr complements a preleukemic defect in a long terminal repeat variant of Moloney murine leukemia virus
Authors: Li Q X , Fan H
Published: January 2020

We previously described a preleukemic state induced by Moloney murine leukemia virus (Mo-MuLV) characterized by hematopoietic hyperplasia in the spleen. Further experiments suggested that splenic hyperplasia results from inhibitory effects in the bone marrow, leading to compensatory extramedullary hematopoiesis. An enhancer variant of Mo-MuLV, Mo + PyF101 Mo-MuLV, fails to induce preleukemic hyperplasia and has greatly reduced leukemogenicity, indicating the importance of this state to efficient leukemogenesis. An alternative method for induction of preleukemic hyperplasia was sought. Treatment of mice with 89Sr causes specific ablation of bone marrow hematopoiesis and compensatory extramedullary hematopoiesis in spleen and nodes. NIH Swiss mice were inoculated neonatally with Mo + PyF101 Mo-MuLV and treated with 89Sr at 6 weeks of age. Approximately 85% developed lymphoid leukemia with a time course resembling that caused by wild-type Mo-MuLV. In contrast, very few animals treated with Mo + PyF101 Mo-MuLV or 89Sr alone developed disease. In approximately one-third of cases, the Mo + PyF101 Mo-MuLV proviruses were found at common sites for wild-type Mo-MuLV-induced tumors (c-myc, pvt-1, and pim-1), indicating that this virus is capable of performing insertional activation in T-lymphoid cells. These results support the proposal that splenic hyperplasia results from inhibitory effects in the bone marrow. They also indicate that Mo + PyF101 Mo-MuLV is blocked in early and not late events in leukemogenesis.
Bacterially expressed nucleoprotein of infectious hematopoietic necrosis virus augments protective immunity induced by the glycoprotein vaccine in fish
Authors: Oberg L A , Wirkkula J , Mourich D , Leong J C
Published: January 2020

The ribonucleoprotein gene of infectious hematopoietic necrosis virus (IHNV) has been expressed in Escherichia coli as a trpE fusion protein. This viral protein does not induce protective immunity to lethal IHNV infection in fish, and virus-neutralizing antibodies do not react with this viral protein. However, when it was administered with a bacterial lysate containing a region of the IHNV glycoprotein, there was enhanced resistance in immunized fish to lethal virus infection.
Identification of a uniquely immunodominant, cross-reacting site in the human immunodeficiency virus endonuclease protein
Authors: Björling E , Utter G , Stålhandske P , Norrby E , Chiodi F
Published: January 2020

One of the features of the life cycle of retroviruses is insertion of the proviral DNA into host chromosomes. A protein encoded by the 3' end of the pol gene of the virus genome has been shown to possess endonuclease activity (D. P. Grandgenett, A. C. Vora, and R. D. Schiff, Virology 89:119-132, 1978), which is necessary for DNA integration. Sera from the majority of human immunodeficiency virus (HIV)-infected individuals react with endonuclease protein p31 in serological tests (J. S. Allan, J. E. Coligan, T.-H. Lee, F. Barin, P. J. Kanki, S. M'Boup, M. F. McLane, J. E. Groopman, and M. Essex, Blood 69:331-333, 1987; E. F. Lillehoj, F. H. R. Salazar, R. J. Mervis, M. G. Raum, H. W. Chan, N. Ahmad, and S. Venkatesan, J. Virol. 62:3053-3058, 1988; K. S. Steimer, K. W. Higgins, M. A. Powers, J. C. Stephans, A. Gyenes, G. George-Nascimento, P. A. Liciw, P. J. Barr, R. A. Hallewell, and R. Sanchez-Pescador, J. Virol. 58:9-16, 1986). It is not known, however, which part of the protein represents the target(s) for antibody response. To study this, we synthesized peptides and used them in an enzyme-linked immunosorbent assay system to map the reactivity of human immunodeficiency virus type 1 (HIV-1) antibody-positive sera to the different regions of the HIV endonuclease. A uniquely antigenic, HIV-1- and HIV-2-cross-reacting site was identified in the central part of this protein from Phe-663 to Trp-670.
Linker insertion mutations in the adenovirus preterminal protein that affect DNA replication activity in vivo and in vitro
Authors: Fredman J N , Pettit S C , Horwitz M S , Engler J A
Published: January 2020

Eighteen linker insertion mutants with mutations in the adenovirus precursor to terminal protein (pTP), which were originally constructed and tested in virions by Freimuth and Ginsberg (Proc. Natl. Acad. Sci. USA 83:7816-7820, 1986), were transferred to expression plasmids for assay of the various functions of the isolated pTP. Function was measured by the ability of individual pTP mutant proteins to participate in the initiation of replication from an adenovirus DNA end, by their activity in assays of DNA elongation, and by the intracellular distribution of pTP demonstrated by indirect immunofluorescence. Ten of the 11 mutants that were active in virion formation were also functional in DNA replication reactions in extracts, while 1 had reduced function. Four mutants with mutations that were lethal to virus production were also inactive in DNA replication reactions. These four mutations are probably located at sites required for the function of pTP in DNA synthesis. Three pTP mutants with mutations that were lethal or partially defective with respect to virion formation were active in reactions requiring pTP for initiation and elongation in extracts. All three of these mutant pTPs targeted normally to the nucleus, suggesting a defect after this step in replication. Since pTP has been reported to bind the nuclear matrix, these pTP mutants may have mutations that define sites necessary for binding to this structure. Several mutants with mutations that lie outside the putative nuclear targeting region were aberrantly localized, suggesting either that additional domains are important in nuclear localization or that there are alterations in protein structure that affect nuclear transport for some pTP mutants.
Hog cholera virus: molecular composition of virions from a pestivirus
Authors: Thiel H J , Stark R , Weiland E , Rümenapf T , Meyers G
Published: January 2020

Virions from hog cholera virus (HCV), a member of the genus Pestivirus, were analyzed by using specific antibodies. The nucleocapsid protein was found to be a 14-kDa molecule (HCV p14). An equivalent protein could also be demonstrated for virions from another pestivirus, bovine viral diarrhea virus. The HCV envelope is composed of three glycoproteins, HCV gp44/48, gp33, and gp55. All three exist in the form of disulfide-linked dimers in virus-infected cells and in virions; HCV gp44/48 and gp55 each form homodimers, whereas gp55 is also found dimerized with gp33. Such complex covalent interactions between structural glycoproteins have not been described so far for any RNA virus.
The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles
Authors: Winokur P L , McLinden J H , Stapleton J T
Published: January 2020

Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.
Antibody enhancement of respiratory syncytial virus stimulation of leukotriene production by a macrophagelike cell line
Authors: Ananaba G A , Anderson L J
Published: January 2020

The clinical and epidemiologic features of respiratory syncytial virus (RSV) infections suggest that RSV-specific antibody may sometimes contribute to the disease process. Recently, it has been demonstrated that virus-specific antibody can enhance RSV infection of macrophagelike cells in vitro. We evaluated the possibility that antibody might also enhance RSV stimulation of the bronchoactive mediator of inflammation leukotriene C-4 (LTC4)in a macrophagelike cell line, U937. The addition of RSV led to little increase in LTC4 production, but addition of RSV plus anti-RSV antibody increased production to a level similar to that achieved with calcium ionophore, a known stimulator of LTC4 production. The antibody-enhanced increase in LTC4 production occurred rapidly (within 15 min), peaked at 60 min, and achieved levels 1.5- to 3.0-fold above that for cells or cells plus virus. RSV plus anti-RSV antibodies in the form of polyclonal serum, monoclonal antibodies, or F(ab')2 fragments and parainfluenza virus types 1 and 3 plus their respective antibodies all increased LTC4 levels over that for the virus alone. These results demonstrate that antibody plus the corresponding virus or protein can increase leukotriene production. This phenomenon could contribute to diseases, such as RSV bronchiolitis, that appear to be caused by an interaction between the virus (or antigen) and host immunity.
Cooperative binding of EF-1A to the E1A enhancer region mediates synergistic effects on E1A transcription during adenovirus infection
Authors: Bruder J T , Hearing P
Published: January 2020

A cellular nuclear factor, EF-1A, binds to a sequence motif which is repeated in the adenovirus type 5 E1A transcriptional control region. Previous genetic analyses demonstrated that two of these binding sites are predominant functional elements of the E1A enhancer region in vivo. In this report, we demonstrate that the cooperative binding of EF-1A to neighboring sites in the E1A enhancer region results in a synergistic activation of E1A transcription in infected cells.
UV activation of human immunodeficiency virus gene expression in transgenic mice
Authors: Vogel J , Cepeda M , Tschachler E , Napolitano L A , Jay G
Published: January 2020

Human immunodeficiency virus (HIV) infection is associated with a clinical latency of as long as 10 years before the development of disease. One explanation for this delay is the requirement of cofactors such as other DNA or RNA viruses, cytokines critical for immune modulation, or environmental UV light. At least in tissue culture studies, these agents are capable of inducing HIV gene expression in cell lines which either harbor the entire viral genome or contain a reporter gene under the control of the viral long terminal repeat regulatory region. The role of these cofactors in terminating clinical latency and inducing disease has been difficult to ascertain because of the lack of an appropriate animal model. We now report that UV light can markedly induce HIV gene expression in transgenic mice carrying both the cis-acting (long terminal repeat) and trans-acting (the tat gene) elements which are essential for viral transactivation and replication in infected cells. Our finding may explain the clinical observations that cutaneous lesions in HIV-infected individuals are often seen in the sunlight exposed areas of the skin, including the face and neck.
Generation of deletion mutants of simian immunodeficiency virus incapable of proviral integration
Authors: Prakash K , Ranganathan P N , Mettus R , Reddy P , Srinivasan A , Plotkin S
Published: January 2020

Deletion mutants of simian immunodeficiency virus (SIVmac) which were unable to integrate into host cells were generated by removing a portion of the integrase (IN) domain of the pol gene. The resulting plasmid was transfected into HUT-78 and human rhabdomyosarcoma cells. In comparison with the parental plasmid DNA transfected in parallel, the deletion mutant was found to direct efficient production of virus in both cell systems. Viruses derived from wild-type and mutant proviral DNAs were also tested for their relative replicative abilities in HUT-78 and U937 cells, and the kinetics of virus production was found to vary between these two cell systems. Analysis of DNA from infected cell nuclei showed that the deletion mutant lacked the ability to integrate despite being able to produce infectious virus. Using the sensitive polymerase chain reaction technique, we have clearly demonstrated the absence of the IN domain in the deletion mutant after infection and replication in HUT-78 cells. Such mutants might form the basis for the development of an experimental live attenuated vaccine.
Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates
Authors: Moore J P , McKeating J A , Huang Y X , Ashkenazi A , Ho D D
Published: January 2020

Primary isolates of human immunodeficiency virus type 1 (HIV-1) are much less sensitive to neutralization by soluble CD4 (sCD4) and sCD4-immunoglobulin (Ig) chimeras (CD4-IgG) than are HIV-1 strains adapted to growth in cell culture. We demonstrated that there are significant reductions (10- to 30-fold) in the binding of sCD4 and CD4-IgG to intact virions of five primary isolates compared with sCD4-sensitive, cell culture-adapted isolates RF and IIIB. However, soluble envelope glycoproteins (gp120) derived from the primary isolate virions, directly by detergent solubilization or indirectly by recombinant DNA technology, differed in affinity from RF and IIIB gp120 by only one- to threefold. The reduced binding of sCD4 to these primary isolate virions must therefore be a consequence of the tertiary or quaternary structure of the envelope glycoproteins in their native, oligomeric form on the viral surface. In addition, the rate and extent of sCD4-induced gp120 shedding from these primary isolates was lower than that from RF. We suggest that reduced sCD4 binding and increased gp120 retention together account for the relative resistance of these primary isolates to neutralization by sCD4 and CD4-IgG and that virions of different HIV-1 isolates vary both in the mechanism of sCD4 binding and in subsequent conformational changes in their envelope glycoproteins.
Characterization of an R-binding site mediating the R-induced activation of the Epstein-Barr virus BMLF1 promoter
Authors: Gruffat H , Duran N , Buisson M , Wild F , Buckland R , Sergeant A
Published: January 2020

In cells latently infected with Epstein-Barr virus, the switch from latency to productive infection is linked to the expression of two Epstein-Barr virus transcription factors called EB1 and R. R is an enhancer factor, and an R-responsive element (RRE) has been identified in the BMLF1 promoter. In this study, we have used bidirectional deletion mutagenesis to delineate the BMLF1 RRE (RRE-M) to a 44-bp sequence. We also show that R expressed from a recombinant vaccinia virus protects RRE-M against digestion by DNase I. Using mobility shift assays and dimethyl sulfate interferences, we have characterized the contact points between in vitro-translated R and the DNA. R binds in vitro to one site by simultaneously contacting two sequences within the site, which are separated by 8 bp: 5'-catGTCCCtctatcatGGCGCagac-3'. Site-directed mutagenesis of this sequence completely impaired the binding of R in vitro and rendered the BMLF1 promoter nonresponsive to R. The results suggest that the R-inducible BMLF1 enhancer is composed of a single R-binding site, called RRE-M.
The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase-primase, are essential for its function
Authors: Zhu L A , Weller S K
Published: January 2020

The UL5 protein of herpes simplex virus type 1, one component of the viral helicase-primase complex, contains six sequence motifs found in all members of a superfamily of DNA and RNA helicases. Although this superfamily contains more than 20 members ranging from bacteria to mammalian cells and their viruses, the importance of these motifs has not been addressed experimentally for any one of them. In this study, we have examined the functional significance of these six motifs for the UL5 protein through the introduction of site-specific mutations resulting in single amino acid substitutions of the most highly conserved residues within each motif. A transient replication complementation assay was used to test the effect of each mutation on the function of the UL5 protein in viral DNA replication. In this assay, a mutant UL5 protein expressed from an expression clone is used to complement a replication-deficient null mutant with a mutation in the UL5 gene for the amplification of herpes simplex virus origin-containing plasmids. Eight mutations in conserved regions and three similar mutations in nonconserved regions of the UL5 gene were analyzed, and the results indicate that all six conserved motifs are essential to the function of UL5 protein in viral DNA replication; on the other hand, mutations in nonconserved regions are tolerated. These data provide the first direct evidence for the importance of these conserved regions in any member of the superfamily of DNA and RNA helicases. In addition, three motif mutations were introduced into the viral genome, and the phenotypic analyses of these mutants are consistent with results from the transient replication complementation assay. The ability of these three mutant UL5 proteins to form specific interactions with other members of the helicase-primase complex, UL8 and UL52, indicates that the functional domains required for replication activity of UL5 are separable from domains responsible for protein-protein interactions. It is anticipated that this type of structure-function analysis will lead to the identification of protein domains that contribute not only to the enzymatic activities of helicase or primase but also to protein-protein interactions within members of the complex.
Nuclear retention of M1 protein in a temperature-sensitive mutant of influenza (A/WSN/33) virus does not affect nuclear export of viral ribonucleoproteins
Authors: Rey O , Nayak D P
Published: January 2020

We investigated the properties of ts51, an influenza virus (A/WSN/33) temperature-sensitive RNA segment 7 mutant. Nucleotide sequence analysis revealed that ts51 possesses a single nucleotide mutation, T-261----C, in RNA segment 7, resulting in a single amino acid change. Phenylalanine (position 79) in the wild-type M1 protein was substituted by serine in ts51. This mutation was phenotypically characterized by dramatic nuclear accumulation of the M1 protein and interfered with some steps at the late stage of virus replication, possibly affecting the assembly and/or budding of viral particles. However, although M1 protein was retained within the nucleus, export of the newly synthesized viral ribonucleoprotein containing the minus-strand RNA into the cytoplasm was essentially the same at both permissive and nonpermissive temperatures. The roles of M1 in the export of viral ribonucleoproteins from the nucleus into the cytoplasm and in the virus particle assembly process are discussed.
Wild-type equine infectious anemia virus replicates in vivo predominantly in tissue macrophages, not in peripheral blood monocytes
Authors: Sellon D C , Perry S T , Coggins L , Fuller F J
Published: January 2020

In situ hybridization of tissues from two horses infected with the wild-type Wyoming strain of equine infectious anemia virus (EIAV) identified the liver, spleen, lymph nodes, kidney, lung, and adrenal gland as the primary host tissue sites for viral transcription during acute infection. Combined immunohistochemistry, with a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes, and in situ hybridization for viral RNA identified most infected cells as mature tissue macrophages. In contrast, in situ hybridization of adherent peripheral blood mononuclear cells collected from horses on various days during the first 2 weeks postinfection with the Wyoming strain of EIAV failed to detect any viral RNA in these cells. For the two horses described here, serum reverse transcriptase activity correlated directly with the degree of replication detected in tissue macrophages on the day of sacrifice. These results suggest that unlike other lentivirus infections in which mature tissue macrophages accumulate cytoplasmic viral RNA to a high level but fail to produce infectious virions, mature tissue macrophages are the likely primary source of the high titer of viremia present during acute infection with EIAV. No significant posttranscriptional block of viral replication in tissue macrophages appears to occur with EIAV.
Fv-1 restriction and its effects on murine leukemia virus integration in vivo and in vitro
Authors: Pryciak P M , Varmus H E
Published: January 2020

We have investigated the mechanisms by which alleles at the mouse Fv-1 locus restrict replication of murine leukemia viruses. Inhibition of productive infection is closely paralleled by reduced accumulation of integrated proviral DNA as well as by reduced levels of linear viral DNA in a cytoplasmic fraction. Nevertheless, viral DNA is present at nearly normal levels in a nuclear fraction, and total amounts of viral DNA are only mildly affected in restrictive infections, suggesting a block in integration to account for reduced levels of proviral DNA. However, integrase (IN)-dependent trimming of 3' ends of viral DNA occurs normally in vivo during restrictive infections, demonstrating that not all IN-mediated events are prevented in vivo. Furthermore, viral integration complexes present in nuclear extracts of infected restrictive cells are fully competent to integrate their DNA into a heterologous target in vitro. Thus, the Fv-1-dependent activity that restricts integration in vivo may be lost in vitro; alternatively, Fv-1 restriction may prevent a step required for integration in vivo that is bypassed in vitro.
Nuclear localization signals, but not putative leucine zipper motifs, are essential for nuclear transport of hepatitis delta antigen
Authors: Chang M F , Chang S C , Chang C I , Wu K , Kang H Y
Published: January 2020

Hepatitis delta antigen (HDAg) is the only known protein of hepatitis delta virus and was previously shown to localize in the nucleoplasm of infected liver cells. In this study, nuclear localization signals of HDAg were defined by expressing various domains of the antigen in both hepatic and nonhepatic cells as beta-galactosidase fusion proteins. A cytochemical staining assay demonstrated that a domain from amino acid residues 35 to 88 of HDAg was able to facilitate transport to the nucleus of the originally cytoplasm-localized protein beta-galactosidase. Two nuclear localization signals, NLS1 and NLS2, which are similar to those of simian virus 40 T antigen and polyomavirus T antigen, respectively, were identified. Either NLS1 or NLS2 alone was sufficient for the nuclear transport of HDAg. However, a fusion protein (N65Z) containing beta-galactosidase and the N-terminal 65 amino acids of HDAg, containing NLS1, was localized exclusively in the cytoplasm and perinuclear region. A possible hydrophobic subdomain between amino acid residues 50 and 65 may block the function of NLS1. Nevertheless, N65Z could enter the nuclei of transfected cells when it was coexpressed with full-length HDAg. Entry into the nucleus may be mediated by the coiled-coil structure rather than the putative leucine zipper motif located between amino acid residues 35 and 65. The existence of two independent nuclear localization signals may ensure the proper functioning of HDAg in the multiplication of delta virus in the nucleus. In addition, two putative casein kinase II sites (SRSE-5 and SREE-126) that may be important in controlling the rate of nuclear transport were found in HDAg.
trans activation of the tumor necrosis factor alpha promoter by the human T-cell leukemia virus type I Tax1 protein
Authors: Albrecht H , Shakhov A N , Jongeneel C V
Published: January 2020

In a cotransfection assay, the human T-cell leukemia virus type I Tax1 gene product specifically activated transcription from the mouse tumor necrosis factor alpha promoter. The activation patterns of 5' deletion mutants, artificial enhancer constructs, and point mutations in the promoter indicate that the major Tax1-responsive element is a site at position -655 which binds the NF-kappa B/rel and NF-GMa transcription factors.
Characterization of nuclear proteins that bind the EFII enhancer sequence in the Rous sarcoma virus long terminal repeat
Authors: Sears R C , Sealy L
Published: January 2020

The EFII cis element is a 38-bp sequence at the 5' end of the Rous sarcoma virus long terminal repeat, extending from nucleotides -229 to -192 (with respect to the viral transcription start site), which is recognized by sequence-specific DNA-binding proteins in avian fibroblast nuclear extracts (L. Sealy and R. Chalkley, Mol. Cell. Biol. 7:787-798, 1987). We demonstrate that multiple copies of the EFII cis element strongly activate transcription of a reporter gene in vivo. We correlate the region of the EFII cis element which activates transcription in vivo with the in vitro binding site for three nuclear factors, EFIIa, EFIIb, and EFIIc. The sequence motif recognized by EFIIa, -b, and -c is also found in consensus binding sites for members of a rapidly growing family of transcription factors related to the CCAAT/enhancer-binding protein (C/EBP). EFIIa, -b, and -c are present in fibroblast and epithelial cell lines from various species but are much less abundant in differentiated rat liver and kidney cells. The EFIIa binding activity is particularly abundant in an avian B-cell lymphoma line. As judged from molecular weight analysis, cell type distribution, and sequence recognition properties, the EFII factors under study appear to differ from most of the previously described C/EBP-related factors and thus may expand the diversity of the C/EBP family.
Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism
Authors: Chesebro B , Wehrly K , Nishio J , Perryman S
Published: January 2020

Previous experiments indicate that the V3 hypervariable region of the human immunodeficiency virus (HIV) envelope protein influences cell tropism of infection; however, so far no consistent V3 sequence can account for macrophage or T-cell tropism. In these experiments, we studied infectious recombinant HIV clones constructed by using V3 region sequences of HIV isolates from 16 patients to search for sequences associated with cell tropism. Remarkable homology was seen among V3 sequences from macrophage-tropic clones from different patients, and a consensus V3 region sequence for patient-derived macrophage-tropic viruses was identified. In contrast, V3 sequences of T-cell-tropic clones from different patients were highly heterogeneous, and the results suggested that sequence diversity leading to T-cell tropism might be generated independently in each patient. Site-specific mutations identified amino acids at several positions on each side of the GPGR motif at the tip of the V3 loop as important determinants of tropism for T cells and macrophages. However, a wide variety of mutant V3 sequences induced macrophage tropism, as detected in vitro. Therefore, the homogeneity of macrophage-tropic patient isolates appeared to be the result of selection based on a biological advantage in vivo.
a1/EBP: a leucine zipper protein that binds CCAAT/enhancer elements in the avian leukosis virus long terminal repeat enhancer
Authors: Bowers W J , Ruddell A
Published: January 2020

Avian leukosis virus (ALV) induces bursal lymphoma in chickens after integration of proviral long terminal repeat (LTR) enhancer sequences next to the c-myc proto-oncogene. Labile LTR-binding proteins appear to be essential for c-myc hyperexpression, since both LTR-enhanced transcription and the activities of LTR-binding proteins are specifically decreased after inhibition of protein synthesis (A. Ruddell, M. Linial, W. Schubach, and M. Groudine, J. Virol. 62:2728-2735, 1988). This lability is restricted to hematopoietic cells from ALV-susceptible chicken strains, suggesting that the labile proteins play an important role in lymphomagenesis. The major labile activity binding to the a1 LTR region (A. Ruddell, M. Linial, and M. Groudine, Mol. Cell. Biol. 12:5660-5668, 1989) was purified from bursal lymphoma cells by conventional and oligonucleotide affinity chromatography, yielding three proteins of 35, 40, and 42 kDa. More than one of these species binds the a1 LTR region, as judged by gel shift analysis. A gene encoding an a1-binding protein (designated a1/EBP) was cloned by screening a bursal lymphoma cDNA library for fusion proteins binding the a1 LTR site. DNase I footprinting and gel shift assays indicate that the a1/EBP fusion protein binds multiple LTR CCAAT/enhancer elements in a pattern similar to that of the purified B-cell protein. DNA sequence analysis shows that this 2.2-kb cDNA encodes a 209-amino-acid open reading frame containing carboxy-terminal basic and leucine zipper motifs, indicating that a1/EBP encodes a novel member of the leucine zipper family of transcription factors.
Fusion mutants of Newcastle disease virus selected with monoclonal antibodies to the hemagglutinin-neuraminidase
Authors: Iorio R M , Glickman R L
Published: January 2020

The Australia-Victoria (AV) isolate of Newcastle disease virus (NDV) induces fusion from within but not fusion from without. L1, a neuraminidase (NA)-deficient virus derived from AV, has the opposite fusion phenotype from the wild-type virus. It fails to induce the former mode of fusion, but has gained a limited ability to promote the latter. Monoclonal antibodies to antigenic site 23 on the hemagglutinin-neuraminidase (HN) glycoprotein have previously been shown to select variants of the AV isolate that have altered NA activity or receptor-binding affinity. By using an antibody to this site, variants of L1 have been selected. Three of the variants have gained an increased affinity for sialic acid-containing receptors, as evidenced by the resistance of their hemagglutinating activity to the presence of reduced amounts of sialic acid on the surface of chicken erythrocytes. All four variants still have very low levels of NA activity, comparable to that of the parent virus, L1. The alteration in receptor-binding affinity results in a decreased potential for elution from cellular receptors and correlates with an increased ability to promote both modes of fusion. A single amino acid substitution in the HN protein of each variant, responsible for its escape from neutralization, has been identified. These studies identify two HN residues, 193 and 203, at which monoclonal antibody-selected substitution influences the receptor recognition properties of NDV and may influence its ability to promote syncytium formation.
Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen
Authors: Xia Y P , Lai M M
Published: January 2020

Two forms of hepatitis delta antigen (HDAg) have different roles in the replication cycle of hepatitis delta virus (HDV); the small forms trans activates HDV RNA replication, whereas the large form suppresses it but is needed for virion assembly. To understand the mechanism of these regulatory activities, we studied the possible HDAg oligomerization and its role in HDV replication. In this report, we provide direct biochemical evidence for the in vitro and in vivo formation of homodimers and heterodimers between these two HDAg species. By deletion mutagenesis, we showed that this protein interaction is mediated by the leucine zipper-like sequence residing in the N-terminal one-third of HDAg. Furthermore, site-specific mutants with various substitutions on two of the leucine residues in this stretch of sequence had reduced or no ability to form HDAg dimers. Correspondingly, the small HDAg with mutations in the leucine zipper-like sequence had reduced abilities to trans activate HDV RNA replication. Similar mutations on the leucine zipper-like sequence of the large HDAg also resulted in loss of the ability of large HDAg to inhibit HDV RNA replication. The in vivo biological activities of both forms of HDAg (trans activation and trans-dominant inhibition of HDV RNA replication, respectively) correlated with the extent of HDAg oligomerization in vitro. Thus, we conclude that the small HDAg participates in HDV RNA replication as an oligomer form and that the large HDAg inhibits HDV RNA replication as a result of its complex formation with small HDAg. A "black sheep" model for the mechanism of trans-dominant inhibition by the large HDAg is presented.
Coexpression of the simian immunodeficiency virus Env and Rev proteins by a recombinant human adenovirus host range mutant
Authors: Cheng S M , Lee S G , Ronchetti-Blume M , Virk K P , Mizutani S , Eichberg J W , Davis A , Hung P P , Hirsch V M , Chanock R M
Published: January 2020

Recombinant human adenoviruses (Ads) that replicate in the intestinal tract offer a novel, yet practical, means of immunoprophylaxis against a wide variety of viral and bacterial pathogens. For some infectious agents such as human immunodeficiency virus (HIV), the potential for residual infectious material in vaccine preparations must be eliminated. Therefore, recombinant human Ads that express noninfectious HIV or other microbial proteins are attractive vaccine candidates. To test such an approach for HIV, we chose an experimental model of AIDS based on simian immunodeficiency virus (SIV) infection of macaques. Our data demonstrate that the SIV Env gene products are expressed in cultured cells after infection with a recombinant Ad containing both SIV env and rev genes. An E3 deletion vector derived from a mutant of human Ad serotype 5 that efficiently replicates in both human and monkey cells was used to bypass the usual host range restriction of Ad infection. In addition, we show that the SIV rev gene is properly spliced from a single SIV subgenomic DNA fragment and that the Rev protein is expressed in recombinant Ad-SIV-infected human as well as monkey cells. The expression of SIV gene products in suitable live Ad vectors provides an excellent system for studying the regulation of SIV gene expression in cultured cells and evaluating the immunogenicity and protective efficacy of SIV proteins in macaques.
Human immunodeficiency virus type 1 gag-protease fusion proteins are enzymatically active
Authors: Kotler M , Arad G , Hughes S H
Published: January 2020

We have introduced mutations into the region of the genome of human immunodeficiency virus type 1 (HIV-1) that encodes the cleavage sites between the viral protease (PR) and the adjacent upstream region of the polyprotein precursor. Segments containing these mutations were introduced into plasmids, and the retroviral proteins were expressed in Escherichia coli. The mutations prevented cleavage between the PR and the adjacent polypeptide; however, other PR cleavage sites in the polyprotein were cleaved normally, showing that the release of free PR is not a prerequisite for the appropriate processing of HIV-1 precursors.
Restoration of cell surface CD4 expression in human immunodeficiency virus type 1-infected cells by treatment with a Tat antagonist
Authors: Shahabuddin M , Volsky B , Hsu M C , Volsky D J
Published: January 2020

Productive infection of T lymphocytes with human immunodeficiency virus type 1 (HIV-1) is accompanied by a diminution of surface CD4 receptors. Treatment of chronically HIV-1-infected CD4-negative T cells in vitro with the Tat antagonist Ro 5-3335 resulted in a drug dose-dependent decrease in virus protein production and a reciprocal increase in surface CD4 display. The drug-treated cells remained viable, showed significantly reduced levels of the full-length and spliced HIV-1 mRNAs as detected by Northern (RNA) blot hybridization, and maintained integrated HIV-1 DNA. In immunoprecipitation studies with drug-treated cells, the levels of free 55-kDa CD4 protein increased and gp160 complexed with CD4 decreased in amount. These results show for the first time that certain cytopathogenic effects of chronic HIV-1 infection can be reversed by suppressing virus expression.