bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence- and structure-specific determinants in the interaction between the RNA encapsidation signal and reverse transcriptase of avian hepatitis B viruses
Authors: Beck J , Nassal M
Published: January 2020

Hepatitis B viruses (HBVs) replicate by reverse transcription of an RNA intermediate. Packaging of this RNA pregenome into nucleocapsids and replication initiation depend crucially on the interaction of the reverse transcriptase, P protein, with the cis-acting, 5' end-proximal encapsidation signal epsilon. The overall secondary structure is similar in all of the hepadnaviral epsilon signals, with a lower and an upper stem, separated by a bulge, and an apical loop. However, while epsilon is almost perfectly conserved in all mammalian viruses, the epsilon signals of duck HBV (DHBV) and heron HBV (D epsilon and H epsilon, respectively) differ substantially in their upper stem regions, both in primary sequence and in secondary structure; nonetheless, H epsilon interacts productively with DHBV P protein, as shown by its ability to stimulate priming, i.e., the covalent attachment of a deoxynucleoside monophosphate to the protein. In this study, we extensively mutated the variable and the conserved positions in the upper stem of D epsilon and correlated the functional activities of the variant RNAs in a priming assay with secondary structure and physical P protein binding. These data revealed a proper overall structure, with the bulge and certain key residues, e.g., in the loop, being important constraints in protein binding. Many mutations at the evolutionarily variable positions complied with these criteria and yielded priming-competent RNAs. However, most mutants at the conserved positions outside the loop were defective in priming even though they had epsilon-like structures and bound to P protein; conversely, one point mutant in the loop with an apical structure different from those of D epsilon and H epsilon was priming competent. These results suggest that P protein binding can induce differently structured epsilon RNAs to adopt a new, common conformation, and they support an induced-fit model of the epsilon-P interaction in which both components undergo extensive structural alterations during formation of a priming-competent ribonucleoprotein complex.
Biological characterization of human immunodeficiency virus type 1 clones derived from different organs of an AIDS patient by long-range PCR
Authors: Dittmar M T , Simmons G , Donaldson Y , Simmonds P , Clapham P R , Schulz T F , Weiss R A
Published: January 2020

In order to characterize the biological properties of human immunodeficiency virus type 1 (HIV-1) variants from different tissues (peripheral blood mononuclear cells [PBMC], lymph node, spleen, brain, and lung) of one patient, we have chosen long-range PCR to amplify virtually full-length HIV proviruses and to construct replication-competent viruses by adding a patient-specific 5' long terminal repeat. To avoid selection during propagation in CD4+ target cells, we transfected 293 cells and used the supernatants from these cells as challenge viruses for tropism studies after titration on human PBMC. Despite differences in the V3 loop of the major variants found in brain and lung compared to lymphoid tissues all recombinant HIV clones obtained showed identical cell tropism and replicative kinetics. After infection of human PBMC these viruses replicated with similar kinetics, with a slow/low-titer, non-syncytium-inducing phenotype. In contrast to the prediction of macrophage tropism, drawn from the V3 loop sequence, none of these viruses infected monocyte-derived macrophages. The challenge of blood dendritic cells by these recombinant viruses in the presence of tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-4 resulted in a productive infection only after adding stimulated CD4+ T lymphocytes. Therefore, the biological properties of the HIV-1 variants derived from nonlymphoid tissue of this patient did not differ from those of HIV-1 variants from lymphoid tissue with respect to tropism for primary cells such as PBMC, macrophages, and blood dendritic cells.
Characterization of late gene transcripts expressed during vegetative replication of human papillomavirus type 31b
Authors: Ozbun M A , Meyers C
Published: January 2020

Human papillomaviruses (HPVs) are etiologic agents of anogenital cancers. The lack of an efficient in vitro system with which to study the differentiation-dependent viral life cycle has impeded most investigations of viral transcription and gene expression. The CIN-612 clone 9E cell line latently maintains episomal copies of HPV type 31b (HPV31b). The complete replicative life cycle of HPV31b can be studied by using the organotypic (raft) culture system. A number of spliced HPV31b early gene transcripts and two late gene transcripts have been described in studies using the raft system. An HPV31b early promoter, P97, and a differentiation-induced promoter, P742, have been characterized by using this system. In this study, we used the raft system to analyze the temporal expression patterns of HPV31b late gene transcripts during the viral life cycle. The expression of late RNAs peaked at day 12 after lifting to the air-liquid interface; the levels then declined dramatically by day 16. The peak of late RNA expression was coincident with the appearance of virus particles in the raft tissues. We characterized transcripts with the potential to encode late gene products, including 19 RNAs containing the L1 region and 4 RNAs containing the E5b and L2 open reading frames. We also found evidence for two novel promoters. Transcription of both L1- and L2-containing RNAs initiated at a region upstream of the early promoter. In addition, late gene RNAs were also transcribed by using a promoter in the E4 reading frame.
Adenovirus vector-infected cells can escape adenovirus antigen-specific cytotoxic T-lymphocyte killing in vivo
Authors: Wadsworth S C , Zhou H , Smith A E , Kaplan J M
Published: January 2020

The recent findings that prolonged expression of certain adenovirus (Ad) vector-encoded proteins, including human alpha1-antitrypsin (huAAT), mouse erythropoietin (EPO), and human factor IX, can be achieved in animals that do not mount an immune response to the reporter protein were obtained with mouse strains which have been shown to be capable of mounting a cellular immune response against Ad vector antigens. This suggests either that Ad vectors expressing nonimmunogenic transgenes fail to elicit a cellular immune response or that an Ad-specific cellular immune response does develop but is ineffective against cells expressing nonimmunogenic transgenes. Here we demonstrate that an Ad vector expressing huAAT administered by intravenous injection does stimulate an Ad-specific cellular immune response but that this response fails to abolish vector-directed gene expression in vivo. Moreover, expression of huAAT remained stable in animals stimulated by concurrent and multiple administrations of different Ad vectors or viruses. We also demonstrate prolonged expression of huAAT in CD1 mice transgenic for the huAAT gene, indicating that long-term expression is not restricted to C57BL/6 mice. These results demonstrate that under some circumstances, an Ad vector can direct prolonged expression of a nonimmunogenic transgene despite the presence of a robust Ad-specific cellular immune response.
Hemagglutinin-esterase, a novel structural protein of torovirus
Authors: Cornelissen L A , Wierda C M , van der Meer F J , Herrewegh A A , Horzinek M C , Egberink H F , de Groot R J
Published: January 2020

We have characterized the 3'-most 3 kb of the genome of bovine torovirus (BoTV) strain Breda. A novel 1.2-kb gene, located between the genes for the membrane and nucleocapsid proteins, was identified. This gene, the 3'-most 0.5 kb of which is also present in the genome of the equine torovirus isolate Berne virus (BEV), codes for a class I membrane protein displaying 30% sequence identity with the hemagglutinin-esterases (HEs) of coronaviruses and influenza C viruses. Heterologous expression of the BoTV HE gene yielded a 65,000-molecular weight N-glycosylated protein displaying acetylesterase activity. Serologic evidence indicates that the HE homolog is expressed during the natural infection and represents a prominent antigen. By using an antiserum raised against residues 13 to 130 of HE, the HE protein was detected in radioiodinated, sucrose gradient-purified BoTV preparations. Formal evidence that HE is a structural protein was provided by immunoelectron microscopy. In addition to the large, 17- to 20-nm spikes, BoTV virions possess shorter surface projections (6 nm on average). We postulate that these surface projections, which are absent from the BEV virion, are composed of the BoTV HE homolog. The HE gene, which has now been demonstrated in three different virus genera, is a showpiece example of modular evolution.
cis-Acting sequences in addition to donor and acceptor sites are required for template switching during synthesis of plus-strand DNA for duck hepatitis B virus
Authors: Havert M B , Loeb D D
Published: January 2020

A characteristic of all hepadnaviruses is the relaxed-circular conformation of the DNA genome within an infectious virion. Synthesis of the relaxed-circular genome by reverse transcription requires three template switches. These template switches, as for the template switches or strand transfers of other reverse-transcribing genetic elements, require repeated sequences (the donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. The mechanism for each of the template switches in hepadnaviruses is poorly understood. To determine whether sequences other than the donor and acceptor sites are involved in the template switches of duck hepatitis B virus (DHBV), a series of molecular clones which express viral genomes bearing deletion mutations were analyzed. We found that three regions of the DHBV genome, which are distinct from the donor and acceptor sites, are required for the synthesis of relaxed-circular DNA. One region, located near the 3' end of the minus-strand template, is required for the template switch that circularizes the genome. The other two regions, located in the middle of the genome and near DR2, appear to be required for plus-strand primer translocation. We speculate that these cis-acting sequences may play a role in the organization of the minus-strand DNA template within the capsid particle so that it supports efficient template switching during plus-strand DNA synthesis.
The small envelope protein is required for secretion of a naturally occurring hepatitis B virus mutant with pre-S1 deleted
Authors: Melegari M , Scaglioni P P , Wands J R
Published: January 2020

Naturally occurring deletions in the hepatitis B virus pre-S1 domain have been frequently found during persistent viral infection. In this study we have investigated the functional properties of a mutant viral genome that carries an in-frame deletion of 183 nucleotides in the pre-S1 region. This deletion removes the promoter of the small envelope gene. Transfection into human hepatocellular carcinoma cells of a replication-competent construct containing this deletion resulted in an increase of intermediate DNA replicative forms compared to those produced by wild-type hepatitis B virus. Northern blot analysis revealed that such cells lack the 2.1-kb transcripts encoding the small envelope protein and that hepatitis B surface antigen was absent as well. Furthermore, nucleocapsids containing the genome with pre-S1 deleted were not secreted, and the deleted large envelope protein was retained with the cytoplasm and exhibited a perinuclear pattern of distribution. However, coexpression with the small envelope protein was sufficient to restore virion secretion and to change the cellular distribution of the deleted large envelope protein. In addition, the creation of point mutations that prevent the synthesis of large or small envelope proteins also inhibited viral secretion and led to increased levels of hepatitis B virus intermediate replicative forms within the cell. These studies suggest that naturally occurring viral mutants with pre-S1 deletions involving the promoter region of the small envelope gene will generate a deleted large envelope protein that is retained in the endoplasmic reticulum, resulting in the accumulation of nucleocapsids containing viral DNA; transcomplementation with the wild-type small envelope protein will allow mutant virion secretion to occur.
Simian virus 40 T antigen can regulate p53-mediated transcription independent of binding p53
Authors: Rushton J J , Jiang D , Srinivasan A , Pipas J M , Robbins P D
Published: January 2020

A simian virus 40 (SV40) T-antigen mutant containing only the N-terminal 136 amino acids, able to bind to Rb and p300 but not p53, partially inhibited p53-mediated transcription without affecting the ability of p53 to bind DNA. These results suggest that SV40 T antigen can regulate p53-mediated transcription either directly through protein-protein association or indirectly through interaction with factors which may function to confer p53-mediated transcription.
Identification of an ATPase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229E
Authors: Heusipp G , Harms U , Siddell S G , Ziebuhr J
Published: January 2020

Human coronavirus 229E gene expression involves proteolytic processing of the gene 1-encoded polyproteins pp1a and pp1ab. In this study, we have detected a 71-kDa polypeptide in virus-infected cells that is released from pp1ab by the virus-encoded 3C-like proteinase and that has been predicted to contain both metal-binding and helicase domains. The polypeptide encompasses amino acids Ala-4996 to Gln-5592 of pp1ab and exhibits nucleic acid-stimulated ATPase activity when expressed as a fusion protein with the Escherichia coli maltose-binding protein. These data provide the first identification of a coronavirus open reading frame 1b-encoded enzymatic activity.
Human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1- and NF-kappaB-binding sites of the interleukin-8 gene
Authors: Murayama T , Ohara Y , Obuchi M , Khabar K S , Higashi H , Mukaida N , Matsushima K
Published: January 2020

Cytomegalovirus (CMV) infection induced interleukin-8 (IL-8) gene transcription in a human monocytic cell line, THP-1 cells, leading to IL-8 secretion. The functional analysis of the IL-8 gene revealed that both AP-1- and NF-kappaB factor-binding elements were involved in conferring the responsiveness to CMV. Moreover, electrophoretic mobility shift assays demonstrated that CMV induced the formation of NF-kappaB and AP-1 complexes. These results suggest that CMV activates these transcriptional factors, resulting in IL-8 gene expression.
Mechanism of human immunodeficiency virus type 1 localization in CD4-negative thymocytes: differentiation from a CD4-positive precursor allows productive infection
Authors: Kitchen S G , Uittenbogaart C H , Zack J A
Published: January 2020

Human immunodeficiency virus (HIV) infection of the thymus could have profound effects on development of the immune response, particularly in children. We and others have established that in addition to infecting and depleting CD4-bearing thymocytes, functional HIV proviruses are found in thymocytes lacking surface CD4 expression. Using in vitro thymocyte cultures, we show that neither HIV-mediated down regulation of CD4 nor CD4-independent infection contributes to the localization of HIV in cells lacking the primary virus receptor. Rather, infection of a CD4-positive precursor cell (CD4 positive/CD8 positive) with subsequent differentiation into a mature CD4-negative phenotype results in productively infected CD4-negative cells. This novel mechanism may contribute to pathogenesis by distributing viral sequences into functional subsets of T cells typically refractory to HIV infection and could account for the presence of viral DNA in CD8-positive lymphocytes recently observed in patients.
Selection of a nonconsensus branch point is influenced by an RNA stem-loop structure and is important to confer stability to the herpes simplex virus 2-kilobase latency-associated transcript
Authors: Krummenacher C , Zabolotny J M , Fraser N W
Published: January 2020

Herpes simplex virus type 1 latent infection in sensory neurons is characterized by the highly restricted transcription of viral genes. The latency-associated transcripts (LAT) family members are the only transcripts that can be identified in large amounts in latently infected cells. The most abundant LAT species is a 2-kb RNA that results from splicing of a rare primary transcript. Analysis of a LAT mutant virus (TB1) in cell culture revealed an aberrant splicing pattern and production of a stable small (0.95-kb) LAT intron. A panel of deletion constructs expressing truncated LAT in transiently transfected cells mapped the region influencing stability to the 3' end of the LAT intron. This region encompasses the branch point and a putative stable stem-loop hairpin structure immediately upstream of the splice acceptor consensus polypyrimidine tract. Mutagenic analysis of the sequence in this region confirmed our hypothesis that the stem-loop structure is important for efficient splicing by influencing the selection of a nonconsensus branch point. Changes in this structure correlate with changes in branch point selection and production of an unstable 2-kb LAT.
Both conserved region 1 (CR1) and CR2 of the human papillomavirus type 16 E7 oncogene are required for induction of epidermal hyperplasia and tumor formation in transgenic mice
Authors: Gulliver G A , Herber R L , Liem A , Lambert P F
Published: January 2020

High-risk human papillomavirus type 16 (HPV-16) and HPV-18 are associated with the majority of human cervical carcinomas, and two viral genes, HPV E6 and E7, are commonly found to be expressed in these cancers. The presence of HPV-16 E7 is sufficient to induce epidermal hyperplasia and epithelial tumors in transgenic mice. In this study, we have performed experiments in transgenic mice to determine which domains of E7 contribute to these in vivo properties. The human keratin 14 promoter was used to direct expression of mutant E7 genes to stratified squamous epithelia in mice. The E7 mutants chosen had either an in-frame deletion in the conserved region 2 (CR2) domain, which is required for binding of the retinoblastoma tumor suppressor protein (pRb) and pRb-like proteins, or an in-frame deletion in the E7 CR1 domain. The CR1 domain contributes to cellular transformation at a level other than pRb binding. Four lines of animals transgenic for an HPV-16 E7 harboring a CR1 deletion and five lines harboring a CR2 deletion were generated and were observed for overt and histological phenotypes. A detailed time course analysis was performed to monitor acute effects of wild-type versus mutant E7 on the epidermis, a site of high-level expression. In the transgenic mice with the wild-type E7 gene, age-dependent expression of HPV-16 E7 correlated with the severity of epidermal hyperplasia. Similar age-dependent patterns of expression of the mutant E7 genes failed to result in any phenotypes. In addition, the transgenic mice with a mutant E7 gene did not develop tumors. These experiments indicate that binding and inactivation of pRb and pRb-like proteins through the CR2 domain of E7 are necessary for induction of epidermal hyperplasia and carcinogenesis in mouse skin and also suggest a role for the CR1 domain in the induction of these phenotypes through as-yet-uncharacterized mechanisms.
The role of the pseudoknot at the 3' end of turnip yellow mosaic virus RNA in minus-strand synthesis by the viral RNA-dependent RNA polymerase
Authors: Deiman B A , Kortlever R M , Pleij C W
Published: January 2020

The tRNA-like structure at the 3' end of turnip yellow mosaic virus (TYMV) RNA was studied in order to determine the role of this structure in the initiation of minus-strand synthesis in vitro. Deletions in the 5'-to-3' direction up to the pseudoknot structure did not result in a decrease of transcription efficiency. However, transcription efficiency was reduced twofold when a fragment of 21 nucleotides, comprising the 3'-terminal hairpin, was used as a template. tRNA(Phe) from yeast, Escherichia coli 5S rRNA, and the 3'-terminal 208 nucleotides of alfalfa mosaic virus RNA 3 could not be transcribed by the RNA-dependent RNA polymerase (RdRp) of TYMV. Various mutations in the sequences of loop regions L1 and L2 or of stem region S1 of the pseudoknot were tested to further investigate the importance of the pseudoknot structure. The results were compared with those obtained in an earlier study on aminoacylation with the same mutants (R. M. W. Mans, M. H. van Steeg, P. W. G. Verlaan, C. W. A. Pleij, and L. Bosch, J. Mol. Biol. 223:221-232; 1992). Mutants which still harbor a stable pseudoknot, as proven by probing its structure, have a transcription efficiency very close to that of the wild-type virus. Disruption of the pseudoknot structure, however, gives rise to a drop in transcription efficiency to about 50%. No indications of base-specific interactions between L1, L2, or S1 of the pseudoknot and the RdRp were found.
The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3
Authors: Osman T A , Buck K W
Published: January 2020

A sucrose density gradient-purified, membrane-bound tobacco mosaic virus (tomato strain L) (TMV-L) RNA polymerase containing endogenous RNA template was efficiently solubilized with sodium taurodeoxycholate. Solubilization resulted in an increase in the synthesis of positive-strand, 6.4-kb genome-length single-stranded RNA (ssRNA) and a decrease in the production of 6.4-kbp double-stranded RNA (dsRNA) to levels close to the limits of detection. The solubilized TMV-L RNA polymerase was purified by chromatography on columns of DEAE-Bio-Gel and High Q. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed that purified RNA polymerase preparations consistently contained proteins with molecular masses of 183, 126, 56, 54, and 50 kDa, which were not found in equivalent material from healthy plants. Western blotting showed that the two largest of these proteins are the TMV-L-encoded 183- and 126-kDa replication proteins and that the 56-kDa protein is related to the 54.6-kDa GCD10 protein, the RNA-binding subunit of yeast eIF-3. The 126-, 183-, and 56-kDa proteins were coimmunoaffinity selected by antibodies against the TMV-L 126-kDa protein and by antibodies against the GCD10 protein. Antibody-linked polymerase assays showed that active TMV-L RNA polymerase bound to antibodies against the TMV-L 126-kDa protein and to antibodies against the GCD10 protein. Synthesis of genome-length ssRNA and dsRNA by a template-dependent, membrane-bound RNA polymerase was inhibited by antibodies against the GCD10 protein, and this inhibition was reversed by prior addition of GCD10 protein.
Truncation of the C-terminal acidic transcriptional activation domain of herpes simplex virus VP16 produces a phenotype similar to that of the in1814 linker insertion mutation
Authors: Smiley J R , Duncan J
Published: January 2020

We examined the phenotype of a herpes simplex virus (HSV) type 1 mutant (V422) in which the C-terminal acidic activation domain of the virion transactivator VP16 is truncated at residue 422. The efficiency of plaque formation by V422 on Vero cells was boosted by approximately 100-fold by including hexamethylene bis-acetimide (HMBA) in the growth medium, as previously observed with the in1814 VP16 linker insertion mutant isolated by Preston and colleagues. V422 displayed severely reduced levels of the immediate-early transcripts encoding ICP0 and ICP4 during infection in the presence of cycloheximide, and this defect was partially overcome by the addition of HMBA. The defect in plaque formation exhibited by V422 and in 1814 was efficiently complemented in U2OS osteosarcoma cells, which had previously been shown to complement ICP0 null mutations. Taken in combination, these data confirm the key role of VP16 in triggering the onset of the HSV lytic cycle.
Interaction of the UV-damaged DNA-binding protein with hepatitis B virus X protein is conserved among mammalian hepadnaviruses and restricted to transactivation-proficient X-insertion mutants
Authors: Sitterlin D , Lee T H , Prigent S , Tiollais P , Butel J S , Transy C
Published: January 2020

We carried out a comparative analysis of several proposed host protein partners of the human hepatitis B virus X protein (HBx) using both the GAL4- and the LexA-based yeast two-hybrid system. We showed that the interaction of HBx with the UV-damaged DNA-binding protein (UVDDB) is positive in both yeast systems, detectable in cotransfected human cells, conserved by rodent hepadnavirus X proteins (known to transactivate in human cells), and tightly correlated with the transactivation proficiency of X-insertion mutants. Taken together, our results strongly suggest that UVDDB is involved in X-mediated transactivation.
Psi- vectors: murine leukemia virus-based self-inactivating and self-activating retroviral vectors
Authors: Delviks K A , Hu W S , Pathak V K
Published: January 2020

We have developed murine leukemia virus (MLV)-based self-inactivating and self-activating vectors to show that the previously demonstrated high-frequency direct repeat deletions are not unique to spleen necrosis virus (SNV) or the neomycin drug resistance gene. Retroviral vectors pKD-HTTK and pKD-HTpTK containing direct repeats composed of segments of the herpes simplex virus type 1 thymidine kinase (HTK) gene were constructed; in pKD-HTpTK, the direct repeat flanked the MLV packaging signal. The generation of hypoxanthine-aminopterin-thymidine-resistant colonies after one cycle of retroviral replication demonstrated functional reconstitution of the HTK gene. Quantitative Southern analysis indicated that direct repeat deletions occurred in 57 and 91% of the KD-HTTK and KD-HTpTK proviruses, respectively. These results demonstrate that (i) deletion of direct repeats occurs at similar high frequencies in SNV and MLV vectors, (ii) MLV psi can be efficiently deleted by using direct repeats, (iii) suicide genes can be functionally reconstituted during reverse transcription, and (iv) the psi region may be a hot spot for reverse transcriptase template switching events.
Transcriptional strategy of closteroviruses: mapping the 5' termini of the citrus tristeza virus subgenomic RNAs
Authors: Karasev A V , Hilf M E , Garnsey S M , Dawson W O
Published: January 2020

Citrus tristeza virus (CTV) induces formation of a nested set of at least nine 3' coterminal subgenomic RNAs (sgRNAs) in infected tissue. The organization and expression of the 19,296-nucleotide (nt) CTV genome resembles that of coronaviruses, with polyprotein processing, translational frameshifting, and multiple sgRNA formation, but phylogenetically the CTV polymerase, like polymerases of other closteroviruses, belongs to the Sindbis virus-like lineage of RNA virus polymerases. Both positive-strand RNA virus supergroups, coronaviruses and Sindbis-like viruses, utilize different mechanisms of transcription. To address the mechanism of CTV transcription, 5' termini for the two most abundant sgRNAs, 1.5 and 0.9 kb, respectively, were mapped by runoff reverse transcription. The two sgRNAs were demonstrated to have 48- and 38-nt 5' untranslated regions (5'-UTRs), respectively. The 5'-UTR for the 1.5-kb RNA was cloned, sequenced, and demonstrated to be colinear with the 48-nt genomic sequence upstream of the initiator codon of the respective open reading frame 10, i.e., to be of continuous template origin. The data obtained suggest that the sgRNA transcription of CTV is dissimilar from the coronavirus transcription and consistent with the transcriptional mechanism of other Sindbis-like viruses. Thus, the Sindbis virus-like mechanism of transcription of the positive-strand RNA genomes might be successfully utilized by the closterovirus genome of up to 19.3 kb with multiple sgRNAs.
RNAs from genetically distinct retroviruses can copackage and exchange genetic information in vivo
Authors: Yin P D , Hu W S
Published: January 2020

Sequence analysis suggests that ancient recombination events may have occurred between genetically distinct retroviruses. An experimental system was utilized to explore the genetic interaction between different viruses. Moloney murine sarcoma virus and spleen necrosis virus are type C retroviruses that belong to different subgenera. With vectors containing packaging signals from these two viruses, DNA proviruses containing genetic information from both RNAs can be generated. This is the first experimental evidence to indicate that RNA from different retroviruses can copackage and exchange genetic information.
A point mutation abolishes the helicase but not the nucleoside triphosphatase activity of hepatitis C virus NS3 protein
Authors: Heilek G M , Peterson M G
Published: January 2020

The NS3 protein of hepatitis C virus contains a bipartite structure consisting of an N-terminal serine protease and a C-terminal DEAD box helicase. We show that the C-terminal domain has ATPase and panhelicase activities. The integrity of the helicase function is dependent on the conserved DEAD motif and can be abolished by a His-Ala point mutation, leaving a fully functional nucleoside triphosphatase.
Evolution of the human immunodeficiency virus type 1 subtype-specific V3 domain is confined to a sequence space with a fixed distance to the subtype consensus
Authors: Lukashov V V , Goudsmit J
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) strains can be separated into genetic subtypes based on phylogenetic analysis of the envelope gene. Once it had been shown that population-wide intrasubtype genetic variation of HIV-1 strains increases in the course of the AIDS epidemic, it remained uncertain whether HIV-1 subtypes are phenotypic entities spreading as distinct virus populations. To examine this, we applied Eigen's concepts of sequence geometry and fitness topography to the analysis of intrasubtype evolution of the gp120 V3 domain of HIV-1 subtypes A, B, C, and D in the course of the global AIDS epidemic. We observed that despite the high evolution rate of HIV-1, the nonsynonymous distances to the subtype consensus of sequences obtained early in the epidemic are similar to those obtained more than 10 years later, in contrast to the synonymous distances, which increased steadily over time. For HIV-1 subtype B, we observed that the evolution rate of the individual sequences is independent of their distance from the subtype B consensus, but for the individual sequences most distant from the consensus evolution away from the consensus is constrained. As a result, individual HIV-1 genomes fluctuate within a sequence space with fixed distance to the subtype consensus. Our findings suggest that the evolution of the V3 domain of HIV-1 subtypes A, B, C, and D is confined to an area in sequence space within a fixed distance to the consensus of a respective subtype. This in turn indicates that each HIV-1 subtype is a distinct viral quasispecies that is well adapted to the present environment, able to maintain its identity in the V3 region over time, and unlikely to merge during progression of the AIDS epidemic.
Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr
Authors: Mahalingam S , Ayyavoo V , Patel M , Kieber-Emmons T , Weiner D B
Published: January 2020

The vpr gene product of human immunodeficiency virus type 1 (HIV-1) is a virion-associated protein that is essential for efficient viral replication in monocytes/macrophages. Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Vpr is packaged efficiently into viral particles through interactions with the p6 domain of the Gag precursor polyprotein p55gag. We developed a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal helical domain, leucine-isoleucine (LR) domain, and carboxy-terminal domain to map the different functional domains and to define the interrelationships between virion incorporation, nuclear localization, cell cycle arrest, and differentiation functions of Vpr. We observed that substitution mutations in the N-terminal domain of Vpr impaired both nuclear localization and virion packaging, suggesting that the helical structure may play a vital role in modulating both of these biological properties. The LR domain was found to be involved in the nuclear localization of Vpr. In contrast, cell cycle arrest appears to be largely controlled by the C-terminal domain of Vpr. The LR and C-terminal domains do not appear to be essential for virion incorporation of Vpr. Interestingly, we found that two Vpr mutants harboring single amino acid substitutions (A30L and G75A) retained the ability to translocate to the nucleus but were impaired in the cell cycle arrest function. In contrast, mutation of Leu68 to Ser resulted in a protein that localizes in the cytoplasm while retaining the ability to arrest host cell proliferation. We speculate that the nuclear localization and cell cycle arrest functions of Vpr are not interrelated and that these functions are mediated by separable putative functional domains of Vpr.
Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses
Authors: Willett B J , Picard L , Hosie M J , Turner J D , Adema K , Clapham P R
Published: January 2020

Feline immunodeficiency virus (FIV) induces a disease state in the domestic cat that is similar to AIDS in human immunodeficiency virus (HIV)-infected individuals. As with HIV, FIV can be divided into primary and cell culture-adapted isolates. Adaptation of FIV to replicate and form syncytia in the Crandell feline kidney (CrFK) cell line is accompanied by an increase in the net charge of the V3 loop of the envelope glycoprotein, mirroring the changes observed in the V3 loop of HIV gp120 with the switch from a non-syncytium-inducing phenotype to a syncytium-inducing phenotype. These data suggest a common mechanism of infection with FIV and HIV. In this study, we demonstrate that cell culture-adapted strains of FIV are able to use the alpha-chemokine receptor CXCR4 for cell fusion. Following ectopic expression of human CXCR4 on nonpermissive human cells, the cells are able to fuse with FIV-infected feline cells. Moreover, fusion between FIV-infected feline cells and CXCR4-transfected human cells is inhibited by both anti-CXCR4 and anti-FIV antibodies. cDNAs encoding the feline CXCR4 homolog were cloned from both T-lymphoblastoid and kidney cell lines. Feline CXCR4 displayed 94.9% amino acid sequence identity with human CXCR4 and was found to be expressed widely on cell lines susceptible to infection with cell culture-adapted strains FIV. Ectopic expression of feline CXCR4 on human cells rendered the cells susceptible to FIV-dependent fusion. Moreover, feline CXCR4 was found to be as efficient as human CXCR4 in supporting cell fusion between CD4-expressing murine fibroblast cells and either HIV type 1 (HIV-1) or HIV-2 Env-expressing human cells. Previous studies have demonstrated that feline cells expressing human CD4 are not susceptible to infection with HIV-1; therefore, further restrictions to HIV-1 Env-dependent fusion may exist in feline cells. As feline and human CXCR4 support both FIV- and HIV-dependent cell fusion, these results suggest a close evolutionary link between FIV and HIV and a common mechanism of infection involving an interaction between the virus and a member of the seven-transmembrane domain chemokine receptor family of molecules.
Mapping promoter regions that are hypersensitive to methylation-mediated inhibition of transcription: application of the methylation cassette assay to the Epstein-Barr virus major latency promoter
Authors: Robertson K D , Ambinder R F
Published: January 2020

Methylation-associated transcriptional repression is recognized in many settings and may play a role in normal differentiation and in tumorigenesis. Both sequence-specific and nonspecific mechanisms have been elaborated. Recently, we have presented evidence that methylation-associated inhibition of the Epstein-Barr virus (EBV) major latency promoter (BamHI C promoter or Cp) in Burkitt's lymphoma and Hodgkin's disease may play an important role in the pathogenesis of these tumors by protecting them from CD8+ cytotoxic T-cell immunosurveillance. The mechanism of transcriptional repression may relate to specific inhibition of the binding of a cellular transcription factor by methylation. To dissect the viral promoter with regard to transcriptional sensitivity to methylation, we have devised an assay that allows the methylation of discrete regions of reporter plasmids. During the course of the assay, methylation patterns appeared to be stable; there was no evidence of either spread or reversal of the imposed methylation pattern. Application of the assay to the 3.8-kb region upstream of the major EBV latency promoter with natural Cp reporter plasmids showed that sensitivity to methylation is not homogeneously distributed but is concentrated in two discrete regions. The first of these methylation-hypersensitive regions (MHRI) is the previously identified EBNA-2 response element, which includes the methylation-sensitive CBF2 binding site. The second (MHRII) is a sequence further downstream whose potential role in methylation-mediated transcriptional repression had been previously unsuspected. In chimeric enhancer/promoter plasmids, methylation of this downstream region was sufficient to virtually abolish simian virus 40 enhancer-driven transcription. Further dissection indicated that methylation of the EBNA-2 response element (MHRI) was sufficient to abolish EBNA-2-mediated Cp activity while methylation of a region including the EBNA-2 response element and downstream sequence (MHRI and MHRII) was sufficient to abolish all Cp-mediated reporter activity, including that driven by the EBNA-1-dependent enhancer in the origin of plasmid replication, oriP.
Inhibition of human immunodeficiency virus type 1 replication in vitro by a novel combination of anti-Tat single-chain intrabodies and NF-kappa B antagonists
Authors: Mhashilkar A M , Biswas D K , LaVecchio J , Pardee A B , Marasco W A
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) Tat, an early regulatory protein that is critical for viral gene expression and replication, transactivates the HIV-1 long terminal repeat (LTR) via its binding to the transactivation response element (TAR) and, along with other cellular factors, increases viral transcription initiation and elongation. Tat also superactivates the HIV-1 promoter through a TAR-independent mechanism, including tumor necrosis factor alpha-induced and protein kinase C (PKC)-dependent activation of NF-kappa B, and inhibitors of Tat and NF-kappa B cooperatively down-regulate this Tat-mediated LTR superactivation. In this study, a combined pharmacologic and genetic strategy using two PKC (NF-kappa B) inhibitors, pentoxifylline (PTX) and Gö-6976, and a stably expressed anti-Tat single-chain intracellular antibody (sFv intrabody) was employed to obtain cooperative inhibition of both HIV-1 LTR-driven gene expression and HIV-1 replication. Treatment of cells with PTX and Gö-6976 resulted in cooperative inhibition of both HIV-1 LTR-driven gene expression and HIV-1 replication. In addition, the combined use of anti-Tat sFv intrabodies and the two NF-kappa B inhibitors retained the virus in the latent state for as long as 45 days. The combined treatment resulted in more durable inhibition of HIV-1 replication than was seen with the NF-kappa B inhibitors alone or the anti-Tat sFv intrabodies alone. Together, these results suggest that in future clinical gene therapy trials, a combined pharmacologic and genetic strategy like the one reported here may improve the survival of transduced cells and prolong clinical benefit.
Minimal truncation of the c-myb gene product in rapid-onset B-cell lymphoma
Authors: Jiang W , Kanter M R , Dunkel I , Ramsay R G , Beemon K L , Hayward W S
Published: January 2020

Oncogenic activation of c-myb by insertional mutagenesis has been implicated in rapid-onset B-cell lymphomas induced by the nonacute avian leukosis virus EU-8. In these tumors, proviruses are integrated either upstream of the c-myb coding region or within the first intron of c-myb. Tumors with either type of integration contained identical chimeric mRNAs in which the viral 5' splice site was juxtaposed to the 3' splice site of c-myb exon 2 and myb exon 1 was eliminated. Both classes of integrations generated truncated Myb proteins that were indistinguishable by Western analysis. In contrast to most other examples of c-myb activation, the truncation consisted of only 20 N-terminal amino acids and did not disrupt either the DNA binding domain near the N terminus or the negative regulatory domain near the C terminus of Myb. The significance of the 20-amino-acid Myb truncation to tumorigenesis was tested by infection of chicken embryos with retroviral vectors expressing different myb gene products. While virus expressing either wild-type c-myb or c-myb mutated at the N-terminal casein kinase II sites was only weakly oncogenic at 10 weeks, the minimally truncated myb virus induced a high incidence of rapid-onset tumors, including B-cell lymphomas, sarcomas, and adenocarcinomas.
Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism
Authors: Spearman P , Horton R , Ratner L , Kuli-Zade I
Published: January 2020

The interaction of the human immunodeficiency virus (HIV) Gag protein with the plasma membrane of a cell is a critical event in the assembly of HIV particles. The matrix protein region (MA) of HIV type 1 (HIV-1) Pr55Gag has previously been demonstrated to confer membrane-binding properties on the precursor polyprotein. Both the myristic acid moiety and additional determinants within MA are essential for plasma membrane binding and subsequent particle formation. In this study, we demonstrated the myristylation-dependent membrane interaction of MA in an in vivo membrane-binding assay. When expressed within mammalian cells, MA was found both in association with cellular membranes and in a membrane-free form. In contrast, the intact precursor Pr55Gag molecule analyzed in an identical manner was found almost exclusively bound to membranes. Both membrane-bound and membrane-free forms of MA were myristylated and phosphorylated. Differential membrane binding was not due to the formation of multimers, as dimeric and trimeric forms of MA were also found in both membrane-bound and membrane-free fractions. To define the requirements for membrane binding of MA, we analyzed the membrane binding of a series of MA deletion mutants. Surprisingly, deletions within alpha-helical regions forming the globular head of MA led to a dramatic increase in overall membrane binding. The stability of the MA-membrane interaction was not affected by these deletions, and no deletion eliminated membrane binding of the molecule. These results establish that myristic acid is a primary determinant of the stability of the Gag protein-membrane interaction and provide support for the hypothesis that a significant proportion of HIV-1 MA molecules may adopt a conformation in which myristic acid is hidden and unavailable for membrane interaction.
The amino-terminal fusion domain peptide of human immunodeficiency virus type 1 gp41 inserts into the sodium dodecyl sulfate micelle primarily as a helix with a conserved glycine at the micelle-water interface
Authors: Chang D K , Cheng S F , Chien W J
Published: January 2020

A peptide based on the N-terminal fusion domain of gp41 of human immunodeficiency virus type 1 (HIV-1) and its tryptophan analog were synthesized to examine the secondary structure in the micellar environment. Nuclear magnetic resonance (NMR), circular dichroism and electron paramagnetic resonance experiments indicated that the gp41 fusion peptide inserted into the micelle primarily as a helix (59%), with substantial beta-structure (26.7%). Deep penetration of the peptide into the apolar hydrocarbon core was supported by the results of fluorescence experiments in which the tryptophan analog exhibited a blue shift of about 30 nm in the presence of a sodium dodecyl sulfate micelle, in 1,2-dimyristoyl-rac-glycero-3-phosphocholine, and in 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine vesicular solutions. The results of spin label-attenuated 1H resonance experiments show that the region C-terminal to G16, which contains a turn structure, exhibited substantial interaction with the micelle, suggesting that it lies on the surface of micelle. Molecular simulation based on data from NMR experiments revealed a flexible hinge at residues 15 and 16 (alanine and glycine, respectively) from the N terminus of the peptide located at the micelle-solution interface. The highly conserved A15-G16 dipeptide may play a role in the function of fusion domain of HIV-1 envelope glycoprotein.
Neuraminidase hemadsorption activity, conserved in avian influenza A viruses, does not influence viral replication in ducks
Authors: Kobasa D , Rodgers M E , Wells K , Kawaoka Y
Published: January 2020

The N1 and N9 neuraminidase (NA) subtypes of influenza A viruses exhibit significant hemadsorption activity that localizes to a site distinct from that of the enzymatic active site. To determine the conservation of hemadsorption activity among different NAs, we have examined most of the NA subtypes from avian, swine, equine, and human virus isolates. All subtypes of avian virus NAs examined and one equine virus N8 NA possessed high levels of hemadsorption activity. A swine virus N1 NA exhibited only weak hemadsorption activity, while in human virus N1 and N2 NAs, the activity was detected at a much lower level than in avian virus NAs. NAs which possessed hemadsorption activity for chicken erythrocytes (RBCs) were similarly able to adsorb human RBCs. However, none of the hemadsorption-positive NAs could bind equine, swine, or bovine RBCs, suggesting that RBCs from these species lack molecules, recognized by the NA hemadsorption site, present on human and chicken RBCs. Mutagenesis of the putative hemadsorption site of A/duck/Hong Kong/7/75 N2 NA abolished the high level of hemadsorption activity exhibited by the wild-type protein but also resulted in a 50% reduction of the NA enzymatic activity. A transfectant virus, generated by reverse genetics, containing this mutated NA replicated 10-fold less efficiently in chicken embryo fibroblast cultures than did a transfectant virus expressing the wild-type NA. However, both viruses replicated equally well in Peking ducks. Although conservation of NA hemadsorption activity among avian virus NAs suggests the maintenance of a required function of NA, loss of the activity does not preclude the replication of the virus in an avian host.