bluuebunny/crossref_metadata_2025 huggingface dataset:
Transient replication of human papillomavirus DNAs
Authors: Del Vecchio A M , Romanczuk H , Howley P M , Baker C C
Published: January 2020

Information on papillomavirus DNA replication has primarily derived from studies with bovine papillomavirus type 1 (BPV-1). Our knowledge of DNA replication of the human papillomaviruses (HPVs) is quite limited, in part because of the lack of a cell culture system capable of supporting the stable replication of HPV DNA. This study demonstrates that the full-length genomic DNAs of HPV types 11 and 18 (HPV-11 and HPV-18), but not HPV-16, are able to replicate transiently after transfection into several different human squamous cell carcinoma cell lines. This system was used to identify the viral cis and trans elements required for DNA replication. The viral origins of replication were localized to a region of the viral long control region. Like BPV-1, E1 and E2 were the only viral factors required in trans for the replication of plasmids containing the origin. Cotransfection of a plasmid expressing the E1 open reading frame (ORF) from HPV-11 with a plasmid that expresses the E2 ORF from HPV-6, HPV-11, HPV-16, or HPV-18 supported the replication of plasmid DNAs containing the origin regions of HPV-11, HPV-16, or HPV-18, indicating that there are functions shared among the corresponding E1 and E2 proteins and origins of these viruses. Although HPV-16 genomic DNA did not replicate by itself under experimental conditions that supported the replication of HPV-11 and HPV-18 genomic DNAs, expression of the HPV-16 early region functions from a strong heterologous promoter supported the replication of a cotransfected plasmid containing the HPV-16 origin of replication. This finding suggests that the inability of the HPV-16 genomic DNA to replicate transiently in the cell lines tested was most likely due to insufficient expression of the viral E1 and/or E2 genes required for DNA replication.
Worldwide prevalence of lentivirus infection in wild feline species: epidemiologic and phylogenetic aspects
Authors: Olmsted R A , Langley R , Roelke M E , Goeken R M , Adger-Johnson D , Goff J P , Albert J P , Packer C , Laurenson M K , Caro T M
Published: January 2020

The natural occurrence of lentiviruses closely related to feline immunodeficiency virus (FIV) in nondomestic felid species is shown here to be worldwide. Cross-reactive antibodies to FIV were common in several free-ranging populations of large cats, including East African lions and cheetahs of the Serengeti ecosystem and in puma (also called cougar or mountain lion) populations throughout North America. Infectious puma lentivirus (PLV) was isolated from several Florida panthers, a severely endangered relict puma subspecies inhabiting the Big Cypress Swamp and Everglades ecosystems in southern Florida. Phylogenetic analysis of PLV genomic sequences from disparate geographic isolates revealed appreciable divergence from domestic cat FIV sequences as well as between PLV sequences found in different North American locales. The level of sequence divergence between PLV and FIV was greater than the level of divergence between human and certain simian immunodeficiency viruses, suggesting that the transmission of FIV between feline species is infrequent and parallels in time the emergence of HIV from simian ancestors.
Mutational analysis of the proposed FG loop of poliovirus proteinase 3C identifies amino acids that are necessary for 3CD cleavage and might be determinants of a function distinct from proteolytic activity
Authors: Hämmerle T , Molla A , Wimmer E
Published: January 2020

Mutations were introduced into a cDNA clone of poliovirus resulting in single-amino-acid substitutions within the region of the proposed FG loop of proteinase 3C. RNAs were made by in vitro transcription with T7 RNA polymerase and used to transfect HeLa cells. Virus viability was assessed as indicated by cell lysis. In parallel, RNAs were translated in vitro by using a HeLa cell lysate, and the patterns of the processed poly-proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Replacement of Lys-78, Arg-79, and Glu-81 had apparently no effect on virus viability and on proteolytic processing. In contrast, virus viability was abolished by mutation of Phe-83, Arg-84, Asp-85, Ile-86, and Arg-87. With respect to substitution of Phe-83, Asp-85, and Arg-87, these effects correlated with impaired processing of the 3CD cleavage site, separating 3C and 3D, and, to a lesser extent, of the P1 precursor. Replacement of Arg-84 and Ile-86, on the other hand, did not alter the processing pattern. Thus, the lethal effects in these mutant genomes may not have been caused by impaired processing. A special case was the mutant of Lys-82-Gln. Virus recovered from cells transfected with RNA carrying this mutation always contained an A-to-G transition which resulted in the replacement of glutamine for arginine. Our data suggest that residues in the proposed FG loop of proteinase 3C influence 3CD cleavage and that they are determinants of a function unrelated to proteolytic processing.
Adenovirus L1 52- and 55-kilodalton proteins are present within assembling virions and colocalize with nuclear structures distinct from replication centers
Authors: Hasson T B , Ornelles D A , Shenk T
Published: January 2020

Analysis of a temperature-sensitive mutant, Ad5ts369, had indicated that the adenovirus L1 52- and 55-kDa proteins (52/55-kDa proteins) are required for the assembly of infectious virions. By using monoclonal antibodies directed against bacterially produced L1 52-kDa protein, the L1 52/55-kDa proteins were found to be differentially phosphorylated forms of a single 48-kDa polypeptide. Both phosphoforms were shown to be present within all suspected virus assembly intermediates (empty capsids, 50 to 100 molecules; young virions, 1 to 2 molecules) but not within mature virions. The mobilities of these proteins in polyacrylamide gels were affected by reducing agents, indicating that the 52/55-kDa proteins may exist as homodimers within the cell and within assembling particles. Immunofluorescence analysis revealed that the 52/55-kDa proteins localize to regions within the infected nucleus that are distinct from viral DNA replication centers, indicating that replication and assembly of viral components likely occur in separate nuclear compartments. Immunoelectron microscopic studies determined that the 52/55-kDa proteins are found in close association with structures that appear to contain assembling virions. These results are consistent with an active but transient role for the L1 products in assembly of the adenovirus particle, perhaps as scaffolding proteins.
Protein P4 of the bacteriophage phi 6 procapsid has a nucleoside triphosphate-binding site with associated nucleoside triphosphate phosphohydrolase activity
Authors: Gottlieb P , Strassman J , Mindich L
Published: January 2020

Bacteriophage phi 6 contains three segments of double-stranded RNA. The procapsid consists of proteins P1, P2, P4, and P7, which are encoded by the viral L segment. cDNA copies of this segment have been cloned into plasmids that direct the production of these proteins, which assemble into polyhedral procapsids. These procapsids are capable of packaging plus-sense phi 6 RNA in the presence of nucleoside triphosphate and synthesizing the complementary minus strand to form double-stranded RNA. In this article, we report the presence of a nucleotide-binding site in protein P4. The viral procapsid and nucleocapsid exhibit a nucleoside triphosphate phosphohydrolase activity that converts nucleoside triphosphates into nucleoside diphosphates.
Surface lysine and tyrosine residues are required for interaction of the major herpes simplex virus type 1 DNA-binding protein with single-stranded DNA
Authors: Ruyechan W T , Olson J W
Published: January 2020

Modification of the herpes simplex virus type 1 major DNA-binding protein (ICP8) with reagents and conditions specific for arginine, lysine, and tyrosine residues indicates that surface lysine and tyrosine residues are required for the interaction of this protein with single-stranded DNA. Modification of either of these two amino acids resulted in a loss and/or modification of binding activity as judged by nitrocellulose filter assays and gel shift. Modification specific for arginine residues did not affect binding within the limits of the assays used. Finally, quenching of the intrinsic tryptophan fluorescence of ICP8 in the presence of single-stranded DNA either suggests involvement of this amino acid in the binding reaction or reflects a conformational change in the protein upon binding.
Mutagenic analysis of the coronavirus intergenic consensus sequence
Authors: Joo M , Makino S
Published: January 2020

Previously, a system in which an intergenic region from mouse hepatitis virus (MHV) inserted into an MHV defective interfering (DI) RNA led to transcription of a subgenomic DI RNA in helper virus-infected cells was established. In the present study, a DI cDNA containing one UCUAAAC consensus sequence in the middle of the 0.3-kb-long intergenic region located between genes 6 and 7 was constructed. From this DI cDNA clone, 21 mutant DI RNAs were constructed so that each of the seven consensus sequence nucleotides was changed individually to the three alternative bases. These mutants were used to define how changes in the integrity of MHV transcription consensus sequence UCUAAAC affected mRNA transcription. Except for two mutants with the sequences UGUAAAC and UCGAAAC, all of the mutants supported efficient subgenomic DI RNA transcription. This indicated that MHV transcription regulation was sufficiently flexible to recognize altered consensus sequences. Next, these and other mutants were used to examine the leader-body fusion site on the subgenomic DI RNAs. Sequence analysis demonstrated that all subgenomic DI RNAs analyzed contained two pentanucleotide sequences; the first sequence seemed to be contributed by the leader, and the leader-body fusion most likely took place at either the first or the second nucleotide of the second sequence. This observation was not consistent with the proposed coronavirus transcription model (S. C. Baker and M. M. C. Lai, EMBO J. 9:4173-4179, 1990) which states that nucleotide mismatch can be corrected by RNA polymerase proofreading activity.
Genetic analysis of simian virus 40 from brains and kidneys of macaque monkeys
Authors: Ilyinskii P O , Daniel M D , Horvath C J , Desrosiers R C
Published: January 2020

Simian virus 40 (SV40) was isolated from the brains of three rhesus monkeys and the kidneys of two other rhesus monkeys with simian immunodeficiency virus-induced immunodeficiency. A striking feature of these five cases was the tissue specificity of the SV40 replication. SV40 was also isolated from the kidney of a Taiwanese rock macaque with immunodeficiency probably caused by type D retrovirus infection. Multiple full-length clones were derived from all six fresh SV40 isolates, and two separate regions of their genomes were sequenced: the origin (ori)-enhancer region and the coding region for the carboxy terminus of T antigen (T-ag). None of the 23 clones analyzed had two 72-bp enhancer elements as are present in the commonly used laboratory strain 776 of SV40; 22 of these 23 clones were identical in their ori-enhancer sequences, and these had only a single 72-bp enhancer element. We found no evidence for differences in ori-enhancer sequences associated with tissue-specific SV40 replication. The T-ag coding sequence that was analyzed was identical in all clones from kidney. However, significant variation was observed in the carboxy-terminal region of T-ag in SV40 isolated from brain tissues. This sequence variation was located in a region previously reported to be responsible for SV40 host range in cultured cell lines. Thus, SV40 appears to be an opportunistic pathogen in the setting of simian immunodeficiency virus-induced immunodeficiency, similarly to JC virus in human immunodeficiency virus-infected humans, the enhancer sequence organization generally attributed to SV40 is not representative of natural SV40 isolates, and sequence variation near the carboxy terminus of T-ag may play a role in tissue-specific replication of SV40.
Requirements for strand transfer between internal regions of heteropolymer templates by human immunodeficiency virus reverse transcriptase
Authors: DeStefano J J , Mallaber L M , Rodriguez-Rodriguez L , Fay P J , Bambara R A
Published: January 2020

We have examined the ability of the reverse transcriptase (RT) from human immunodeficiency virus (HIV) to carry out strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions of natural-sequence RNA. A 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide was used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptors. In this system, HIV RT produced strand transfer products. An HIV RT having RNase H depleted to 3% of normal (HIV RTRD) catalyzed the transfer reaction inefficiently. An RNase H-minus deletion mutant of murine leukemia virus RT was unable to catalyze strand transfer. HIV RTRD, however, efficiently catalyzed transfer when Escherichia coli RNase H was included in the reactions, while the mutant murine leukemia virus RT was not efficiently complemented by the E. coli enzyme. Evidently, RNase H activity enhances, or is required for, internal strand transfer. Two acceptors homologous to 27-nucleotide regions of the donor, one offset from the other by 6 nucleotides, were tested. The offset eliminated a sequence homologous to a prevalent DNA synthesis pause site in the donor. Strand transfer to this acceptor was about 25% less efficient, suggesting that RT pausing can enhance strand transfer. When the deoxynucleoside triphosphates in the reactions were reduced from 50 to 0.2 microM, increasing RT pausing, the efficiency of strand transfer also increased. A model for RT-catalyzed strand transfer consistent with our results is presented.
Persistence of viral RNA in mouse brains after recovery from acute alphavirus encephalitis
Authors: Levine B , Griffin D E
Published: January 2020

Little is known about the relationship between recovery from acute viral encephalitis and the clearance of viral genetic material from the central nervous system. In a mouse model of Sindbis virus encephalitis, we have previously shown that clearance of infectious virus is mediated by antibody-induced restriction of viral gene expression rather than by cytotoxic destruction of virally infected cells. To explore whether Sindbis virus genomes persist in mouse brain after the clearance of infectious virus, we used reverse transcriptase-polymerase chain reaction amplification methods to detect Sindbis virus RNA in brain samples from immunocompetent BALB/c and antibody-treated immunodeficient scid/CB17 mice. RNA sequences from both the nonstructural region (NSP1 gene) and structural regions (E2 gene) of Sindbis virus were detected in the brains of all BALB/c and antibody-treated scid mice examined at 1, 2, and 3 months after infection. Additional BALB/c mouse brains were also positive at 8, 12, and 17 months after infection. To determine whether persistent RNA was capable of resuming unrestricted replication in the absence of the continuous presence of antiviral antibodies, viral titers were measured in the brains of scid mice at 1, 2, 3, and 6 months after antibody treatment. Viral reactivation was seen in scid mice treated with hyperimmune serum or a low dose of monoclonal antibody to the E2 envelope glycoprotein, but not in mice treated with a high dose of monoclonal antibody to E2. Replication of infectious virus isolated from scid mouse brain could be restricted by repeat treatment with immune serum, indicating that viral reactivation is not due to antibody-escape mutations. These results demonstrate that Sindbis virus can persist long term in a nonproductive form in mouse brain and suggest that the humoral immune response plays an important role in preventing viral reactivation.
Maturation cleavage required for infectivity of a nodavirus
Authors: Schneemann A , Zhong W , Gallagher T M , Rueckert R R
Published: January 2020

Nodaviral morphogenesis involves formation of labile precursor particles, called provirions, which mature by autocatalytic cleavage of the 407-residue coat precursor protein between asparagine residue 363 and alanine residue 364. It has previously been demonstrated that maturation results in increased physicochemical stability of the virion. We show here that cleavage of coat protein in purified provirions of Flock House virus was accompanied by a five- to eightfold increase in specific infectivity. Cleavage-negative provirions, produced by site-directed mutagenesis of asparagine residue 363 to aspartate, threonine, or alanine, displayed no infectivity above revertant frequencies as measured by plaque assay. All viable revertants (nine of nine) restored asparagine to the mutated position, suggesting high specificity for asparagine at the cleavage site.
Activation of the prolactin receptor gene by promoter insertion in a Moloney murine leukemia virus-induced rat thymoma
Authors: Barker C S , Bear S E , Keler T , Copeland N G , Gilbert D J , Jenkins N A , Yeung R S , Tsichlis P N
Published: January 2020

The prolactin receptor (Prlr) and growth hormone receptor (Ghr) genes and the Moloney murine leukemia virus integration-2 (Mlvi-2) locus were mapped to mouse chromosome 15 and human chromosome 5 bands p12-p14. To examine the potential relationship between Mlvi-2 and the genes encoding the growth hormone receptor and the prolactin receptor, we determined the chromosomal location of all three loci in the rat, using a panel of rat-mouse somatic cell hybrids, and in the mouse, using a panel of (C57BL/6J x Mus spretus)F1 x C57BL/6J interspecific backcross mice. These analyses revealed that Ghr, Prlr, and Mlvi-2 map to chromosome 2 in the rat and to chromosome 15 in the mouse, in close proximity with each other. Pulsed-field gel electrophoresis of rat genomic DNA showed no overlaps between the gene encoding the prolactin receptor and the remaining loci. Moreover, expression of the prolactin receptor was not affected by provirus insertion in Mlvi-2. During these studies, however, we detected one T-cell lymphoma line (2779) in which the prolactin receptor gene was activated by provirus integration. Sequence analysis of polymerase chain reaction-derived cDNA clones showed that the prolactin receptor RNA message initiates at the 5' long terminal repeat and utilizes the splice donor site 5' of the gag gene to splice the viral sequences onto exon 1 of the prolactin receptor. This message is predicted to encode the intact prolactin receptor protein product. Exposure of the T-cell lymphoma line 2779 to prolactin promoted cellular proliferation.
Minimal requirements for the human immunodeficiency virus type 1 V3 domain to support the syncytium-inducing phenotype: analysis by single amino acid substitution
Authors: De Jong J J , De Ronde A , Keulen W , Tersmette M , Goudsmit J
Published: January 2020

The third variable domain (V3) of the human immunodeficiency virus type 1 external envelope contains determinants of cell tropism, cytopathicity, and infectivity and elicits antibodies able to block infectivity in vitro and in vivo. Our study encompassed point-mutational analysis of HXB-2 viruses containing patient-derived V3 regions and expressing a non-syncytium-inducing, low-replicating phenotype in T-cell line SupT1. The mutation within V3 of a serine at position 306 into an also naturally occurring arginine (S to R) required an additional, naturally occurring mutation at position 320 (aspartate to glutamine, D to Q) or 324 (aspartate to asparagine, D to N) for full expression of the syncytium-inducing, high-replicating (SI) phenotype. The naturally occurring mutation of an aspartate into an arginine at position 320 (D to R) was sufficient for production of the SI phenotype. This study proves that introduction of a positively charged amino acid at position 306 or 320, previously shown to be strongly associated with the SI phenotype in field isolates (R.A.M. Fouchier, M. Groenink, N.A. Kootstra, M. Tersmette, H.G. Huisman, F. Miedema, and H. Schuitemaker, J. Virol. 66:3183-3187, 1992), is minimally required for production of SI viruses. In addition, naturally occurring mutations at residue 324 also modulate the virus phenotype.
Construction and characterization of a human cytomegalovirus mutant with the UL18 (class I homolog) gene deleted
Authors: Browne H , Churcher M , Minson T
Published: January 2020

The UL18 open reading frame of human cytomegalovirus (HCMV) (which encodes a product homologous to major histocompatibility complex class I heavy chains) has been disrupted by insertion of the beta-galactosidase gene under control of the major HCMV early promoter. The recombinant virus delta UL18 showed no phenotypic differences from wild-type HCMV in terms of single-step growth curves or particle/infectivity ratios, indicating that the UL18 gene product is dispensable for the growth of HCMV in human fibroblasts in vitro. The synthesis of the mature cellular class I heterodimer is shut down in cells infected at a high multiplicity with wild-type HCMV, and a similar effect was seen in delta UL18-infected fibroblasts, suggesting that although the UL18 gene product can associate with beta 2 microglobulin, it is not directly involved in the disruption of class I assembly.
Mutational analysis of the major homology region of Mason-Pfizer monkey virus by use of saturation mutagenesis
Authors: Strambio-de-Castillia C , Hunter E
Published: January 2020

The major capsid (CA) protein of retroviruses possesses a stretch of 20 amino acids, called the major homology region (MHR), which is evolutionarily conserved and invariant in location within the primary sequence of the protein. The function of this region was investigated by examining the effect of random single-amino-acid substitutions within the central 13 positions of the MHR on the life cycle of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive D-type retrovirus. When these mutants were subcloned into an M-PMV proviral vector and expressed in COS cells, one of two major phenotypes was observed. The first group, containing three mutants bearing drastic amino acid substitutions, was unable to assemble capsids in the cytoplasm of the host cell. The second and more common group of mutants was able to assemble and release virions, but these either displayed greatly reduced levels of infectivity or were completely noninfectious. Included within this second group were two mutants with unusual phenotypes; mutant D158Y exhibited a novel cleavage site for the viral protease that resulted in cleavage of the major capsid protein, p27 (CA), within the MHR, whereas mutant F156L appeared to have lost a major site for antibody recognition within the mature CA protein. The results of this mutagenic analysis suggest that changes in the MHR sequence can interfere with the assembly of viral capsids and block an early stage of the infection cycle of M-PMV.
Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4
Authors: Willey R L , Maldarelli F , Martin M A , Strebel K
Published: January 2020

CD4 is an integral membrane glycoprotein which is known as the human immunodeficiency virus (HIV) receptor for infection of human cells. The protein is synthesized in the endoplasmic reticulum (ER) and subsequently transported to the cell surface via the Golgi complex. HIV infection of CD4+ cells leads to downmodulation of cell surface CD4, due at least in part to the formation of stable intracellular complexes between CD4 and the HIV type 1 (HIV-1) Env precursor polyprotein gp160. This process "traps" both proteins in the ER, leading to reduced surface expression of CD4 and reduced processing of gp160 to gp120 and gp41. We have recently demonstrated that the presence of the HIV-1-encoded integral membrane protein Vpu can reduce the formation of Env-CD4 complexes, resulting in increased gp160 processing and decreased CD4 stability. We have studied the effect of Vpu on CD4 stability and found that Vpu induces rapid degradation of CD4, reducing the half-life of CD4 from 6 h to 12 min. By using a CD4-binding mutant of gp160, we were able to show that this Vpu-induced degradation of CD4 requires retention of CD4 in the ER, which is normally accomplished through its binding to gp160. The involvement of gp160 in the induction of CD4 degradation is restricted to its function as a CD4 trap, since, in the absence of Env, an ER retention mutant of CD4, as well as wild-type CD4 in cultures treated with brefeldin A, a drug that blocks transport of proteins from the ER, is degraded in the presence of Vpu.
Expression and characterization of the trans-activating protein Tax of human T-cell leukemia virus type I in Saccharomyces cerevisiae
Authors: Wu L C , Tan T H , Shahied S I
Published: January 2020

The trans-activator protein Tax of human T-cell leukemia virus type I (HTLV-I) stimulates transcription of the viral genome from the long terminal repeat. With a reporter HIS4TATA::lacZ fusion gene, the transcriptional activity of the Tax-responsive element in the long terminal repeat was tested in Saccharomyces cerevisiae. We found that fragments containing the 21-bp repeat of the HTLV-I enhancer stimulate synthesis of beta-galactosidase activity 15- to 20-fold. To test the ability of the Tax protein to trans activate the HTLV-I enhancer in yeast cells, the pX region of HTLV-I, encoding the Tax protein, was cloned under the control of the yeast GAL1 promoter. The expressed Tax protein is localized in the nucleus and associated with the yeast nuclear matrix fraction. In yeast cells that contained the integrated tax gene, two- to sixfold stimulation of expression from the HTLV-I enhancer was detected at the early stages of tax induction. This in vivo reconstitution system provides a new approach for examining the host factor(s), the signal transduction mechanism(s), and the role of nuclear architecture involved in Tax-mediated trans activation.
A constitutive enhancer in the bovine papillomavirus upstream regulatory region shares genetic elements with the viral P1 promoter
Authors: Bream G L , Vaillancourt P , Botchan M R
Published: January 2020

The bovine papillomavirus upstream regulatory region represents a common element in the regulation of transcription from the five early viral promoters. We have determined the sequences required for transcription from the viral P1 promoter, which is located at the 5' end of the upstream regulatory region. In vitro transcription from P1 requires a 123-bp fragment (nucleotides 7153 to 7275; -33 to +90) consisting of an upstream TATA-like sequence as well as an unidentified protein which binds to sequences immediately downstream of the initiation site. In vivo, this promoter requires additional downstream sequences (to position +160; nucleotide 7345) for maximal activity but does not require any additional DNA sequence upstream of a putative TATA box. Four regions within the downstream sequence from +9 to +160 are protected from DNase I digestion by proteins present in a HeLa cell extract. The presence of these sites correlates with the level of P1 activity. A constitutive enhancer maps to this same region, and mutations in this enhancer have been shown to affect downstream promoters. Deletion analysis indicates that the same sequences are required by both the P1 promoter and the constitutive enhancer, suggesting that the same proteins function in both activities.
Herpes simplex virus type 1 mutant strain in1814 establishes a unique, slowly progressing infection in SCID mice
Authors: Valyi-Nagy T , Deshmane S L , Raengsakulrach B , Nicosia M , Gesser R M , Wysocka M , Dillner A , Fraser N W
Published: January 2020

Ocular infection of immunocompetent (BALB/c) mice with wild-type herpes simplex virus type 1 (HSV-1) 17+ may lead to acute fatal encephalitis; however, in surviving animals, a latent (nonproductive) infection of the nervous system is established. In contrast, 17+ infection invariably kills mice with severe combined immunodeficiency (SCID mice) within 2 weeks. Ocular infection of immunocompetent mice with a mutant HSV-1 strain, in1814, which does not produce a functional alpha-transinducing protein, results in no detectable viral replication in the nervous system during the time corresponding to the acute phase of infection, no mortality, and the establishment of latency. In SCID mice, however, the in1814 virus establishes a unique, slowly progressing infection. In studying the courses of in1814 infection in SCID and BALB/c mice, we found that although intact B- and/or T-lymphocytic functions were required for the control of viral replication in the nervous system, some of the infected neurons of SCID mice seemed to be able to restrict in1814 replication and harbor the virus in a latent state.
A mouse mammary tumor virus mammary gland enhancer confers tissue-specific but not lactation-dependent expression in transgenic mice
Authors: Mok E , Golovkina T V , Ross S R
Published: January 2020

The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) is known to contain a number of transcriptional regulatory elements, including glucocorticoid response elements. In this study, we showed that a mammary gland/salivary gland enhancer found in the LTR of this virus directs expression of a heterologous promoter to both virgin and lactating mammary glands in transgenic mice. Using transgenic mice containing hybrid gene constructs with various deletions of the LTR sequences linked to marker genes, we also showed that the dramatic increase in MMTV expression that occurs during lactation is due to the glucocorticoid response elements. Thus, the MMTV LTR encodes two distinct elements, both of which are required for a high level of expression in lactating mammary glands.
Effects of altering palmitylation sites on biosynthesis and function of the influenza virus hemagglutinin
Authors: Naim H Y , Amarneh B , Ktistakis N T , Roth M G
Published: January 2020

Mutagenesis studies indicated that the three cytoplasmic cysteines of the influenza virus A/Japan/305/57 hemagglutinin (HA) are all palmitylated, but to an unequal extent. Replacement of all three cysteines abolished palmitylation, but affected neither HA biosynthesis nor function. Palmitate was not required for HA to be incorporated into virions.
Restriction fragment length polymorphism of polymerase chain reaction products from vaccine and wild-type varicella-zoster virus isolates
Authors: LaRussa P , Lungu O , Hardy I , Gershon A , Steinberg S P , Silverstein S
Published: January 2020

The nucleotide changes that result in two restriction endonuclease polymorphisms that differentiate wild-type varicella-zoster virus (VZV) from the vaccine strain were determined. Oligonucleotide primers that flank these sites were used to amplify the intervening sequences with the polymerase chain reaction to identify VZV DNA in clinical isolates. Restriction enzyme digestion of the amplification products distinguished vaccine and wild-type genomes from one another. This study confirms the feasibility of amplifying VZV sequences so that they may be detected in clinical specimens and provides a molecular epidemiological approach to strain identification of VZV in vesicular lesions.
Complex formation of human T-cell leukemia virus type I p40tax transactivator with cellular polypeptides
Authors: Nagata K , Ide Y , Takagi T , Ohtani K , Aoshima M , Tozawa H , Nakamura M , Sugamura K
Published: January 2020

We examined cellular components which associate with p40tax, the viral transactivation molecule of human T-cell leukemia virus type I. Such molecules were searched by immunoprecipitation with polyclonal and monoclonal antibodies specific for p40tax. Two cellular proteins with molecular masses of 95 kDa (p95) and 60 kDa (p60) were specifically coprecipitated with p40tax from extracts of all p40tax-producing cell lines but not from p40tax-negative cell lines. The p60 component was also shown to associate with p40tax in vitro, by using radiolabel-chase experiments. Rabbit antisera specific for p60 and p95 were prepared by immunization with electrophoretically purified molecules. While anti-p95 antiserum coprecipitated p40tax, no p40tax could be identified in immunoprecipitates by using a polyclonal anti-p60 antiserum. The partial amino acid sequence of p60 demonstrated that p60 is identical to the human 60-kDa heat shock protein (a member of the chaperonin family of proteins). Although the biological significance of the complex formation of p40tax with p95 and p60 has yet to be determined, it may be that the complex formation is one of the mechanisms by which the biological activity of p40tax can be regulated.
Sequential phosphorylation of the phosphoprotein of vesicular stomatitis virus by cellular and viral protein kinases is essential for transcription activation
Authors: Barik S , Banerjee A K
Published: January 2020

The phosphoprotein (P) and the large protein (L) constitute the RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV). We show that phosphate-free P protein expressed in bacteria is transcriptionally inactive when reconstituted with L protein and viral N-RNA template free of cellular protein kinase. Phosphorylation of P protein by a cellular kinase(s) was essential for transcription as well as for further phosphorylation by an L-associated kinase, the two kinases acting in a sequential (cascade) manner. Phosphate groups introduced by cell kinase were stable, whereas those due to L kinase underwent a turnover which was coupled to ongoing transcription. We present a model for the phosphorylation pathway of P protein and propose that continued phosphorylation and dephosphorylation of P protein may represent a transcriptional regulatory (on-off) switch of nonsegmented negative-strand RNA viruses.
Vaccination with a synthetic peptide modulates lymphocytic choriomeningitis virus-mediated immunopathology
Authors: Battegay M , Oehen S , Schulz M , Hengartner H , Zinkernagel R M
Published: January 2020

Vaccination with a nucleopeptide (NP 118; amino acids 118 to 132) representing a cytotoxic T-cell epitope of lymphocytic choriomeningitis virus (LCMV) can modulate immunopathology. Immunization with NP 118 protected H-2d mice against intracerebral infection with the LCMV-ARMSTRONG isolate. However, when NP 118-primed H-2d mice were challenged intracerebrally with an intermediate dose (5 x 10(4) PFU) of the LCMV-DOCILE strain, all mice primed with NP 118 emulsified in incomplete Freund's adjuvant died, whereas unprimed mice survived. Correspondingly, peptide vaccination enhanced specifically the cytotoxic T-cell response, influencing the critical balance between T-cell response and virus spread.
Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies
Authors: Connors M , Kulkarni A B , Collins P L , Firestone C Y , Holmes K L , Morse H C , Murphy B R
Published: January 2020

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance.
A cyclic AMP-responsive DNA-binding protein (CREB2) is a cellular transactivator of the bovine leukemia virus long terminal repeat
Authors: Willems L , Kettmann R , Chen G , Portetelle D , Burny A , Derse D
Published: January 2020

To gain insight into the cellular regulation of bovine leukemia virus (BLV) trans activation, a lambda-gt11 cDNA library was constructed with mRNA isolated from a BLV-induced tumor and the recombinant proteins were screened with an oligonucleotide corresponding to the tax activation-responsive element (TAR). Two clones (called TAR-binding protein) were isolated from 750,000 lambda-gt11 plaques. The binding specificity was confirmed by Southwestern (DNA-protein) and gel retardation assays. Nucleotide sequence analysis revealed that TAR-binding protein is very similar to the CREB2 protein. It contains a leucine zipper structure required for dimerization, a basic amino acid domain, and multiple potential phosphorylation sites. A vector expressing CREB2 was transfected into D17 osteosarcoma cells. In the absence of the tax transactivator, the CREB2 protein and the cyclic AMP-dependent protein kinase A activate the BLV long terminal repeat at a basal expression level: trans activation reached 10% of the values obtained in the presence of tax alone. These data demonstrate that CREB2 is a cellular factor able to induce BLV long terminal repeat expression in the absence of tax protein and could thus be involved in the early stages of viral infection. In addition, we observed that in vitro tax-induced trans activation can be activated or inhibited by CREB2 depending on the presence or absence of protein kinase A. These data suggest that the cyclic AMP pathway plays a role in the regulation of viral expression in BLV-infected animals.
Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses
Authors: Bess J W , Powell P J , Issaq H J , Schumack L J , Grimes M K , Henderson L E , Arthur L O
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) were purified by sucrose density gradient centrifugation in the presence of 1 mM EDTA. Pelleted gradient fractions were analyzed for total protein, total Gag capsid protein, and total zinc. Zinc was found to copurify and concentrate with the virus particles. Through successive cycles of resuspending in buffer containing EDTA and repelleting, the zinc content remained constant at about 1.7 mol of zinc per mol of Gag protein. Proteins from purified virus (HIV-1 and HTLV-I) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to polyvinylidene fluoride paper, and probed with 65ZnCl2. Viral nucleocapsid (NC) proteins (HIV-1 p7NC and HTLV-I p15NC) bound 65Zn2+. Other retroviruses, including simian immunodeficiency virus, equine infectious anemia virus, bovine leukemia virus, Moloney murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus, were found to contain amounts of zinc per milligram of total protein similar to those found in HIV-1 and HTLV-I. Collectively, these data support the hypothesis that retroviral NC proteins function as zinc finger proteins in mature viruses.
Identification and characterization of a neutralization site within the second variable region of human immunodeficiency virus type 1 gp120
Authors: Fung M S , Sun C R , Gordon W L , Liou R S , Chang T W , Sun W N , Daar E S , Ho D D
Published: January 2020

Two monoclonal antibodies designated BAT085 and G3-136 were raised by immunizing BALB/c mice with gp120 purified from human immunodeficiency virus type 1 (HIV-1) IIIB-infected H9 cell extracts. Among three HIV-1 laboratory isolates (IIIB, MN, and RF), BAT085 neutralized only IIIB infection of CEM-SS cells, whereas G3-136 neutralized both IIIB and RF. These antibodies also neutralized a few primary HIV-1 isolates in the infection of activated human peripheral blood mononuclear cells. In indirect immunofluorescence assays, BAT085 bound to H9 cells infected with IIIB or MN, while G3-136 bound to H9 cells infected with IIIB or RF, but not MN. Using sequence-overlapping synthetic peptides of HIV-1 IIIB gp120, the binding site of BAT085 and G3-136 was mapped to a peptidic segment in the V2 region (amino acid residues 169 to 183). The binding of these antibodies to immobilized gp120 was not inhibited by the antibodies directed to the principal neutralization determinant in the V3 region or to the CD4-binding domain of gp120. In a competition enzyme-linked immunosorbent assay, soluble CD4 inhibited G3-136 but not BAT085 from binding to gp120. Deglycosylation of gp120 by endo-beta-N-acetylglucosaminidase H or reduction of gp120 by dithiothreitol diminished its reactivity with G3-136 but not with BAT085. These results indicate that the V2 region of gp120 contains multiple neutralization determinants recognized by antibodies in both a conformation-dependent and -independent manner.
Utilization of heterologous alphavirus junction sequences as promoters by Sindbis virus
Authors: Hertz J M , Huang H V
Published: January 2020

We used Sindbis virus, an alphavirus, as a model to study the evolution of the recognition of viral cis-acting sequences. During the life cycle of alphaviruses, a full-length minus-strand RNA is made and serves as a template for both genomic RNA replication and subgenomic mRNA transcription. Transcription initiates at an internal promoter site, the junction sequence, to produce a subgenomic mRNA. The junction sequences of alphaviruses are highly conserved, but they do contain a number of base differences. These could have been essentially neutral mutations during evolution, such that any of the contemporary sequences can be recognized efficiently by any of the alphaviruses. Alternately, the changes could have resulted in significant functional divergence, such that the contemporary viruses can no longer recognize heterologous junction sequences as promoters. To distinguish between these possibilities, we constructed Sindbis virus derivatives with two subgenomic mRNA promoters. One is the wild-type Sindbis virus promoter used for expression of the structural proteins. The other is either the minimal Sindbis virus promoter or the corresponding junction sequences from other alphaviruses, which are placed upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene. RNA analyses were used to determine the relative promoter strengths of the various junction sequences. The results showed that all but two were recognized as promoters by Sindbis virus. CAT enzyme assays were used to measure the accumulation of CAT protein made from mRNAs transcribed by using the heterologous junction sequences as promoters. Most of the viruses expressed amounts of CAT enzyme within 10-fold of each other. The two viruses with junction sequences that were not recognized as promoters did not give significant CAT expression. We conclude that, with respect to Sindbis virus, the junction sequences are functionally conserved; i.e., most of the contemporary nucleotide differences in the junction sequences are neutral or near-neutral mutations. The functional conservation suggests that neither the cis-acting sequence nor the cognate binding site of the transcription factor can change independently. This type of coupled evolution between cis-acting sequences and their cognate viral protein binding sites may be a general phenomenon. For example, it explains the ubiquitous presence of conserved cis-acting sequences in each of the families of RNA viruses. There are implications of this hypothesis for the design of antiviral drugs.
Regions of human papillomavirus type 16 E7 oncoprotein required for immortalization of human keratinocytes
Authors: Jewers R J , Hildebrandt P , Ludlow J W , Kell B , McCance D J
Published: January 2020

Binding of the retinoblastoma gene product (pRB) by viral oncoproteins, including the E7 of human papillomavirus type 16 (HPV 16), is thought to be important in transformation of cells. One of the steps in transformation is the immortalization process. Here we show that mutations in E7 within the full-length genome which inhibit binding of pRB do not abrogate the ability of the HPV 16 DNA to immortalize primary human epithelial (keratinocyte) cells. A mutation in one of the cysteines of a Cys-X-X-Cys motif which is contained in the carboxy half of the E7 and is part of a zinc finger arrangement completely eliminates the ability of HPV 16 DNA to immortalize cells. The results indicate the importance of E7 in the immortalization of primary keratinocytes but suggest that the binding of pRB is not essential.
Unusually high frequency of reconstitution of long terminal repeats in U3-minus retrovirus vectors by DNA recombination or gene conversion
Authors: Olson P , Temin H M , Dornburg R
Published: January 2020

Recently, we described a retrovirus vector system with which to study formation of cDNA genes (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 6:2328-2334, 1988; Mol. Cell. Biol. 8:64-72, 1990; J. Virol. 64:886-889, 1990). For these studies, retrovirus vectors were constructed in which the U3 region of the 3' long terminal repeat (LTR) was deleted. After one round of retrovirus replication, such vectors formed a provirus with two U3-minus LTRs. However, the insertion of some additional sequences into such vectors promoted vector rearrangements with an efficiency greater than 95%. Such rearranged vectors behaved like vectors with two wild-type LTRs. Proviruses derived from such vectors were investigated by Southern blot analysis, polymerase chain reaction, and DNA sequencing. We found that the U3 region was reconstituted, resulting in vectors with LTRs like wild-type virus. The sequences that reconstituted the U3 region of the vector LTR were derived from LTR sequences present in the helper cell. Since no retroviral protein coding sequences were detected in infected target cells, recombination of vector sequences with coencapsidated helper cell sequences during reverse transcription seems very unlikely. Thus, it appears that the recombination (or gene conversion) events leading to a vector with reconstituted LTRs occurred at the DNA level. The high frequency of this recombination (or gene conversion) was dependent on internal vector sequences.