bluuebunny/crossref_metadata_2025 huggingface dataset:
A Hairpin Structure in the R Region of the Human Immunodeficiency Virus Type 1 RNA Genome Is Instrumental in Polyadenylation Site Selection
Authors: Das Atze T. , Klaver Bep , Berkhout Ben
Published: December 2019

Some retroviruses with an extended repeat (R) region encode the polyadenylation signal within the R region such that this signal is present at both the 5′ and 3′ ends of the viral transcript. This necessitates differential regulation to either repress recognition of the 5′ polyadenylation signal or enhance usage of the 3′ signal. The human immunodeficiency virus type 1 (HIV-1) genome encodes an inherently efficient polyadenylation signal within the 97-nucleotide R region. Polyadenylation at the 5′ HIV-1 polyadenylation site is inhibited by downstream splicing signals, and usage of the 3′ polyadenylation site is triggered by an upstream enhancer element. In this paper, we demonstrate that this on-off switch of the HIV-1 polyadenylation signal is controlled by a secondary RNA structure that occludes part of the AAUAAA hexamer motif, which we have termed the polyA hairpin. Opening the 5′ hairpin by mutation triggered premature polyadenylation and caused reduced synthesis of viral RNA, indicating that the RNA structure plays a pivotal role in repression of the 5′ polyadenylation site. Apparently, the same hairpin structure does not interfere with efficient usage of the 3′ polyadenylation site, which may be due to the presence of the upstream enhancer element. However, when the 3′ hairpin was further stabilized by mutation, we measured a complete loss of 3′ polyadenylation. Thus, the thermodynamic stability of the polyA hairpin is delicately balanced to allow nearly complete repression of the 5′ site yet efficient activation of the 3′ site. This is the first report of regulated polyadenylation that is mediated by RNA secondary structure. A similar hairpin motif that occludes the polyadenylation signal can be proposed for other lentiviruses and members of the spumaretroviruses, suggesting that this represents a more general gene expression strategy of complex retroviruses.
Genetic Selection of Poliovirus 2A
pro
-Binding Peptides
Authors: Ventoso Iván , Barco Angel , Carrasco Luis
Published: December 2019

The yeast two-hybrid system has been used to identify mammalian clones that interact with poliovirus 2A proteinase (2A
pro
). Eight clones which encode previously unidentified human proteins were selected from a HeLa cell cDNA expression library. In addition, five clones encoding short peptides that interact with poliovirus 2A
pro
were also identified. The lengths of these peptides range from 6 to 30 amino acids, but all of them contain the Leu-X-Thr-Z motif (X represents any amino acid; Z represents a hydrophobic residue). This sequence is invariably located just at the carboxy terminus of each peptide. This approach raises the possibility of designing substrate analogue inhibitors of 2A
pro
. Thus, two nonhydrolyzable peptides containing the Leu-X-Thr-Z motif prevented cleavage of eukaryotic initiation factor 4G by poliovirus 2A
pro
in vitro. A more general method for identifying peptides with antiproteinase activity is discussed.
Truncated Particles Produced in Fish Surviving Infectious Hematopoietic Necrosis Virus Infection: Mediators of Persistence?
Authors: Kim Carol H. , Dummer Dina M. , Chiou Pinwen P. , Leong Jo-Ann C.
Published: December 2019

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that produces an acute, lethal infection in rainbow trout (
Oncorhynchus mykiss
). Fish that survive infection cease to produce detectable infectious virus at approximately 46 days after infection, yet there is evidence that survivor fish continue to harbor virus particles (B. S. Drolet, P. P. Chiou, J. Heidel, and J. C. Leong, J. Virol. 69:2140–2147, 1995). In an effort to determine the biological function of these particles, the kidneys and livers from IHNV survivors were harvested and divided into samples for nested reverse transcriptase PCR analysis and explant culture. Sequences for the IHNV nucleoprotein and polymerase genes were detected in 50 and 89%, respectively, of the organs from survivor fish. When explant tissue cultures were infected with purified standard IHNV, the liver tissues from survivor fish produced up to 10-fold less virus than naive control fish liver tissues. In addition, immunosorbent electron microscopy analysis of the supernatant media from the cultured explants of survivor fish revealed truncated particles, whereas the control tissue supernatants contained only standard viral particles. These results suggest that the truncated IHNV particles observed in persistently infected fish are defective interfering particles that may mediate virus persistence.
Viral Load and a Locus on Chromosome 11 Affect the Late Clinical Disease Caused by Theiler’s Virus
Authors: Aubagnac Stéphanie , Brahic Michel , Bureau Jean-François
Published: December 2019

Theiler’s virus causes a persistent infection and a demyelinating disease of mice which is a model for multiple sclerosis. Susceptibility to viral persistence maps to several loci, including the interferon gamma locus. Inactivating the gene coding for the interferon gamma receptor makes 129/Sv mice susceptible to persistent infection and clinical disease, whereas inactivating the interferon gamma gene makes C57BL/6 mice susceptible to persistent infection but not to clinical disease. This difference in phenotype is due to the difference in genetic background. Clinical disease depends on high viral load andTmevd5, a locus on chromosome 11. These results have consequences for the identification of viruses which might be implicated in multiple sclerosis.
CD40-Mediated Induction of CD4 and CXCR4 on B Lymphocytes Correlates with Restricted Susceptibility to Human Immunodeficiency Virus Type 1 Infection: Potential Role of B Lymphocytes as a Viral Reservoir
Authors: Moir Susan , Lapointe Réjean , Malaspina Angela , Ostrowski Mario , Cole Charsey E. , Chun Tae-Wook , Adelsberger Joseph , Baseler Michael , Hwu Patrick , Fauci Anthony S.
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) replicates primarily in lymphoid tissues where it has ready access to activated immune competent cells. We used one of the major pathways of immune activation, namely, CD40-CD40L interactions, to study the infectability of B lymphocytes isolated from peripheral blood mononuclear cells. Highly enriched populations of B lymphocytes generated in the presence of interleukin-4 and oligomeric soluble CD40L upregulated costimulatory and activation markers, as well as HIV-1 receptors CD4 and CXCR4, but not CCR5. By using single-round competent luciferase viruses complemented with either amphotropic or HIV-derived envelopes, we found a direct correlation between upregulation of HIV-1 receptors and the susceptibility of the B lymphocytes to infection with dual-tropic and T-tropic strains of HIV-1; in contrast, cells were resistant to M-tropic strains of HIV-1. HIV-1 envelope-mediated infection was completely abolished with either an anti-CD4 monoclonal antibody or a peptide known to directly block CXCR4 usage and partially blocked with stromal cell-derived factor 1, all of which had no effect on the entry of virus pseudotyped with amphotropic envelope. Full virus replication kinetics confirmed that infection depends on CXCR4 usage. Furthermore, productive cycles of virus replication occurred rapidly yet under most conditions, without the appearance of syncytia. Thus, an activated immunological environment may induce the expression of HIV-1 receptors on B lymphocytes, priming them for infection with selective strains of HIV-1 and allowing them to serve as a potential viral reservoir.
Discovery of a New Endogenous Type C Retrovirus (FcEV) in Cats: Evidence for RD-114 Being an FcEV
Gag-Pol
/Baboon Endogenous Virus BaEV
Env
Recombinant
Authors: van der Kuyl Antoinette C. , Dekker John T. , Goudsmit Jaap
Published: December 2019

Analysis of a cat genomic DNA library showed that cats harbor a previously unrecognized endogenous type C retrovirus, whose
env
gene has homology to the murine
Fv-4
resistance gene. This unique retrovirus, designated FcEV (
Felis catus
endogenous retrovirus), has a type C
pol
gene, closely related to the primate
Papio cynocephalus
endogenous virus (PcEV)
pol
, not overlapping the
env
gene, unlike in other type C retroviruses, and is presumably present in a higher copy number than RD-114. Phylogenetic analysis of FcEV and RD-114 fragments amplified from cat species and comparison with baboon endogenous virus (BaEV) fragments from monkeys suggested that RD-114 does not represent the cat strain of BaEV but is actually a new recombinant between FcEV type C genes and the
env
gene of BaEV. Although BaEV did appear to have infected an ancestor of the domestic cat lineage, it was a de novo recombinant that made its way into the cat germ line.
Translation from the 5′ Untranslated Region (UTR) of mRNA 1 Is Repressed, but That from the 5′ UTR of mRNA 7 Is Stimulated in Coronavirus-Infected Cells
Authors: Senanayake Savithra D. , Brian David A.
Published: December 2019

Viral gene products are generally required in widely differing amounts for successful virus growth and assembly. For coronaviruses, regulation of transcription is a major contributor to these differences, but regulation of translation may also be important. Here, we examine the possibility that the 5′ untranslated regions (UTRs), unique for each of the nine species of mRNA in the bovine coronavirus and ranging in length from 70 nucleotides (nt) to 210 nt (inclusive of the common 5′-terminal 65-nt leader), can differentially affect the rate of protein accumulation. When the natural 77-nt 5′ UTR on synthetic transcripts of mRNA 7 (mRNA for N and I proteins) was replaced with the 210-nt 5′ UTR from mRNA 1 (genomic RNA, mRNA for viral polymerase), approximately twofold-less N, or (N) CAT fusion reporter protein, was made in vitro. Twofold less was also made in vivo in uninfected cells when a T7 RNA polymerase-driven transient-transfection system was used. In coronavirus-infected cells, this difference surprisingly became 12-fold as the result of both a stimulated translation from the 77-nt 5′ UTR and a repression of translation from the 210-nt 5′ UTR. These results reveal that a differential 5′ UTR-directed regulation of translation can occur in coronavirus-infected cells and lead us to postulate that the direction and degree of regulation is carried out by viral or virally induced cellular factors acting in
trans
on
cis
-acting elements within the 5′ UTR.
Genetic Reassortment of Rift Valley Fever Virus in Nature
Authors: Sall A. A. , Zanotto P. M. de A. , Sene O. K. , Zeller H. G. , Digoutte J. P. , Thiongane Y. , Bouloy M.
Published: December 2019

Rift Valley fever virus (RVFV), a phlebovirus of the
Bunyaviridae
family, is an arthropod-borne virus which emerges periodically throughout Africa, emphasizing that it poses a major threat for animal and human populations. To assess the genetic variability of RVFV, several isolates from diverse localities of Africa were investigated by means of reverse transcription-PCR followed by direct sequencing of a region of the small (S), medium (M), and large (L) genomic segments. Phylogenetic analysis showed the existence of three major lineages corresponding to geographic variants from West Africa, Egypt, and Central-East Africa. However, incongruences detected between the L, M, and S phylogenies suggested that genetic exchange via reassortment occurred between strains from different lineages. This hypothesis, depicted by parallel phylogenies, was further confirmed by statistical tests. Our findings, which strongly suggest exchanges between strains from areas of endemicity in West and East Africa, strengthen the potential existence of a sylvatic cycle in the tropical rain forest. This also emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated vaccines in areas of endemicity.
Role of Immune Responses against the Envelope and the Core Antigens of Simian Immunodeficiency Virus SIVmne in Protection against Homologous Cloned and Uncloned Virus Challenge in Macaques
Authors: Polacino Patricia S. , Stallard Virginia , Klaniecki James E. , Pennathur Sridhar , Montefiori David C. , Langlois Alphonse J. , Richardson Barbra A. , Morton William R. , Benveniste Raoul E. , Hu Shiu-Lok
Published: December 2019

We previously showed that envelope (gp160)-based vaccines, used in a live recombinant virus priming and subunit protein boosting regimen, protected macaques against intravenous and intrarectal challenges with the homologous simian immunodeficiency virus SIVmne clone E11S. However, the breadth of protection appears to be limited, since the vaccines were only partially effective against intravenous challenge by the uncloned SIVmne. To examine factors that could affect the breadth and the efficacy of this immunization approach, we studied (i) the effect of priming by recombinant vaccinia virus; (ii) the role of surface antigen gp130; and (iii) the role of core antigens (Gag and Pol) in eliciting protective immunity. Results indicate that (i) priming with recombinant vaccinia virus was more effective than subunit antigen in eliciting protective responses; (ii) while both gp130 and gp160 elicited similar levels of SIV-specific antibodies, gp130 was not as effective as gp160 in protection, indicating a possible role for the transmembrane protein in presenting functionally important epitopes; and (iii) although animals immunized with core antigens failed to generate any neutralizing antibody and were infected upon challenge, their virus load was 50- to 100-fold lower than that of the controls, suggesting the importance of cellular immunity or other core-specific immune responses in controlling acute infection. Complete protection against intravenous infection by the pathogenic uncloned SIVmne was achieved by immunization with both the envelope and the core antigens. These results indicate that immune responses to both antigens may contribute to protection and thus argue for the inclusion of multiple antigens in recombinant vaccine designs.
Human Herpesvirus 8 Interleukin-6 (vIL-6) Signals through gp130 but Has Structural and Receptor-Binding Properties Distinct from Those of Human IL-6
Authors: Wan Xiaoyu , Wang Hailin , Nicholas John
Published: December 2019

Human herpesvirus 8 (HHV-8) has been associated with classical, endemic (African), and AIDS-related Kaposi’s sarcoma (KS), body cavity-based primary effusion lymphomas, and multicentric Castleman’s disease (MCD). HHV-8 encodes a functional homologue of interleukin-6 (IL-6), a cytokine that promotes the growth of KS and myeloma cells and is found at elevated levels in MCD lesions and patient sera. We have previously reported that the viral IL-6 (vIL-6) gene product can support the growth of the IL-6-dependent murine hybridoma cell line, B9, and that the gp80 (IL-6 receptor [IL-6R]) component of the IL-6 receptor-signal transducer (gp180) complex plays a role in mediating this activity. However, it has been shown by others that vIL-6 can function in human cells independently of IL-6R. Here we have extended our functional studies of vIL-6 by identifying transcription factors and pathways used in human Hep3B cells, investigating the utilization of gp130 and IL-6R by vIL-6, and undertaking mutational analyses of vIL-6 and gp130. The data presented here establish that vIL-6, in common with its endogenous counterparts, can mediate signal transduction through gp130 and activate multiple transcription factors, map residues within the vIL-6 protein that are and are not important for vIL-6 signalling, and identify a gp130 mutant that is nonfunctional with respect to vIL-6 signalling in the absence of IL-6R but that retains the ability to mediate vIL-6 and human IL-6 (hIL-6) signal transduction when IL-6R is coexpressed. The data presented demonstrate functional and mechanistic similarities between vIL-6 and endogenous IL-6 proteins but also highlight differences in the structural and receptor-binding properties of vIL-6 relative to its human counterpart.
Translational Effects of Mutations and Polymorphisms in a Repressive Upstream Open Reading Frame of the Human Cytomegalovirus UL4 Gene
Authors: Alderete John P. , Jarrahian Sohail , Geballe Adam P.
Published: December 2019

The human cytomegalovirus (HCMV) gpUL4 mRNA contains a 22-codon upstream open reading frame (uORF2), the peptide product of which represses downstream translation by blocking translation termination at its own stop codon and by causing ribosomes to stall on the mRNA. A distinctive feature of this unusual mechanism is its strict dependence on the uORF2 peptide sequence. To delineate sequence elements that function in the inhibitory mechanism, deletions and missense mutations affecting the previously uncharacterized amino-terminal region of uORF2 were analyzed in transient-transfection and infection assays. These experiments identified multiple codons in this region that are necessary for inhibition of downstream translation by uORF2 and, in conjunction with previous results, demonstrated that amino acids dispersed throughout the uORF2 peptide participate in the repressive mechanism. In contrast to the highly conserved carboxy terminus, the amino-terminal portion of the uORF2 peptide is polymorphic. A survey of uORF2 sequences in HCMV clinical isolates revealed that although most have uORF2 sequences that are predicted to retain the uORF2 inhibitory activity, ∼15% contain polymorphisms at codons that are essential for full inhibition by uORF2. Consistent with predictions based on analyses of engineered mutations, two viral isolates with uORF2 sequences that do not inhibit downstream translation in transfection assays expressed much more gpUL4 protein but similar levels of UL4 mRNA compared to the levels produced by the prototypic laboratory strain HCMV (Towne) and another clinical isolate with an inhibitory variant uORF2. These results demonstrate that uORF2 is polymorphic in sequence and repressive activity and suggest that the uORF2 regulatory mechanism, although prevalent among natural HCMV isolates, is not absolutely essential for viral replication.
Human Immunodeficiency Virus Type 1 Gag Polyprotein Multimerization Requires the Nucleocapsid Domain and RNA and Is Promoted by the Capsid-Dimer Interface and the Basic Region of Matrix Protein
Authors: Burniston Mark T. , Cimarelli Andrea , Colgan John , Curtis Sean P. , Luban Jeremy
Published: December 2019

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein directs the formation of virions from productively infected cells. Many
gag
mutations disrupt virion assembly, but little is known about the biochemical effects of many of these mutations. Protein-protein interactions among Gag monomers are believed to be necessary for virion assembly, and data suggest that RNA may modify protein-protein interactions or even serve as a bridge linking Gag polyprotein monomers. To evaluate the primary sequence requirements for HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants was expressed in bacteria and evaluated for the ability to associate with full-length Gag in vitro. The nucleocapsid protein, the major RNA-binding domain of Gag, exhibited activity comparable to that of the complete polyprotein. In the absence of the nucleocapsid protein, relatively weak activity was observed that was dependent upon both the capsid-dimer interface and basic residues within the matrix domain. The relevance of the in vitro findings was confirmed with an assay in which nonmyristylated mutant Gags were assessed for the ability to be incorporated into virions produced by wild-type Gag expressed in
trans
. Evidence of the importance of RNA for Gag-Gag interaction was provided by the demonstration that RNase impairs the Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags encoded by distantly related retroviruses and with structurally unrelated RNA-binding proteins. These results are consistent with models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions.
Mutations within the
Autographa californica
Nucleopolyhedrovirus
FP25K
Gene Decrease the Accumulation of ODV-E66 and Alter Its Intranuclear Transport
Authors: Braunagel Sharon C. , Burks Jared K. , Rosas-Acosta German , Harrison Robert L. , Ma H. , Summers M. D.
Published: December 2019

Previous reports indicate that mutations within the
Autographa californica
nucleopolyhedrosis virus
FP25K
gene (open reading frame 61) significantly reduce incorporation of enveloped nucleocapsids into viral occlusions. We report that FP25K is a nucleocapsid protein of both the budded virus (BV) and occluded virus (ODV), and we describe the effects of two
FP25K
mutations (480-1 [N-terminal truncation] and FP-βgal [C-terminal fusion]) on the expression and cellular localization of ODV-E66 and ODV-E25. Significantly decreased amounts of ODV-E66 are detected in cells infected with 480-1 or FP-βgal viral mutants, even though during FP-βgal infection, steady-state levels of ODV-E66 transcripts remain unchanged. While ODV-E66 is normally detected in intranuclear microvesicles and ODV envelopes by 24 h postinfection (p.i.), ODV-E66 remains cytosolic throughout infection in cells infected with 480-1 virus (up to 96 h p.i.), and its intranuclear localization is not detected until 96 h p.i. in cells infected with the FP-βgal mutant virus. The nuclear localization of ODV-E25 is not affected during infection by the FP-βgal mutant; however, its trafficking is significantly delayed during infection by the 480-1 mutant. Temporal Western blot analyses of cell lysates show that both 480-1 and FP-βgal mutant virus infections result in altered accumulation patterns of several structural proteins, including gp67, BV/ODV-E26, and the major capsid protein p39. In addition to BV/ODV-E26, ODV-E66 and gp67 may interact with FP25K, and ODV-E25 and p39 may also be components of a protein complex containing ODV-E66 and FP25K. Together, these data suggest that FP25K and its associated protein complex(es) may play an important role in the targeting and intracellular transport of viral proteins during infection.
Inhibition of Human Immunodeficiency Virus Type 1 Infectivity by the gp41 Core: Role of a Conserved Hydrophobic Cavity in Membrane Fusion
Authors: Ji Hong , Shu Wei , Burling F. Temple , Jiang Shibo , Lu Min
Published: December 2019

The gp41 envelope protein of human immunodeficiency virus type 1 (HIV-1) contains an α-helical core structure responsible for mediating membrane fusion during viral entry. Recent studies suggest that a conserved hydrophobic cavity in the coiled coil of this core plays a distinctive structural role in maintaining the fusogenic conformation of the gp41 molecule. Here we investigated the importance of this cavity in determining the structure and biological activity of the gp41 core by using the N34(L6)C28 model. The high-resolution crystal structures of N34(L6)C28 of two HIV-1 gp41 fusion-defective mutants reveal that each mutant sequence is accommodated in the six-helix bundle structure by forming the cavity with different sets of atoms. Remarkably, the mutant N34(L6)C28 cores are highly effective inhibitors of HIV-1 infection, with 5- to 16-fold greater activity than the wild-type molecule. The enhanced inhibitory activity by fusion-defective mutations correlates with local structural perturbations close to the cavity that destabilize the six-helix bundle. Taken together, these results indicate that the conserved hydrophobic coiled-coil cavity in the gp41 core is critical for HIV-1 entry and its inhibition and provides a potential antiviral drug target.
In Vitro and In Vivo Characterization of a Mouse Adenovirus Type 1 Early Region 3 Null Mutant
Authors: Cauthen Angela N. , Brown Corrie C. , Spindler Katherine R.
Published: December 2019

Previous attempts to construct a mouse adenovirus type 1 early region 3 (E3) null mutant by initiator codon mutagenesis were unsuccessful because one of the E3 proteins, gp11K, is synthesized as a fusion protein from a late viral mRNA (A. N. Cauthen and K. R. Spindler, Virology 259:119–128, 1999). Therefore, a different mutagenesis strategy was employed that inserted termination codons into all three reading frames of the E3 proteins. This strategy produced a mutant,
pm
E314, that was null for the expression of E3 proteins as determined by immunoprecipitation with E3-specific antisera. This mutant grew as well as wild-type (wt) virus in both 3T6 mouse fibroblasts and mouse brain microvascular endothelial cells. However, the 50% lethal dose for
pm
E314 in adult NIH Swiss outbred mice was approximately 6 log units higher than that of wt virus, indicating that
pm
E314 was less virulent in mice. In situ hybridization experiments revealed that the absence of the E3 proteins did not alter the tropism of the mutant virus from that of wt virus. When the histopathology was evaluated, the characteristics of the
pm
E314 infection at both doses administered were strikingly different from those exhibited by wt virus. The central nervous system of wt-infected mice exhibited damage to the endothelium and recruitment of inflammatory cells, whereas the central nervous system of
pm
E314-infected mice showed no inflammatory response and only mild signs of endothelial damage.
Functional Analysis of the Interaction between VPg-Proteinase (NIa) and RNA Polymerase (NIb) of Tobacco Etch Potyvirus, Using Conditional and Suppressor Mutants
Authors: Daròs José-Antonio , Schaad Mary C. , Carrington James C.
Published: December 2019

The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa). To investigate the significance of this interaction, a
Saccharomyces cerevisiae
two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (
ts
) defects in interacting with NIb. Thirty-six unique
ts
NIa mutants with substitutions affecting the proteinase domain were recovered. Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures. However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (
ts
NIa-Q384P and
ts
NIa-N393D), the mutations responsible for the
ts
interaction phenotype could be mapped to single positions. One of the mutations (N393D) conferred a
ts
-genome-amplification phenotype when it was placed in a recombinant TEV strain. Suppressor NIb mutants that restored interaction with the
ts
NIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system. Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the
ts
NIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold. These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication.
Vaccinia Virus Envelope D8L Protein Binds to Cell Surface Chondroitin Sulfate and Mediates the Adsorption of Intracellular Mature Virions to Cells
Authors: Hsiao Jye-Chian , Chung Che-Sheng , Chang Wen
Published: December 2019

We previously showed that an envelope A27L protein of intracellular mature virions (IMV) of vaccinia virus binds to cell surface heparan sulfate during virus infection. In the present study we identified another viral envelope protein, D8L, that binds to chondroitin sulfate on cells. Soluble D8L protein interferes with the adsorption of wild-type vaccinia virions to cells, indicating a role in virus entry. To explore the interaction of cell surface glycosaminoglycans and vaccinia virus, we generated mutant viruses from a control virus, WR32-7/Ind14K (A27L
+
D8L
+
) to be defective in expression of either the A27L or the D8L gene (A27L
+
D8L
−
or A27L
−
D8L
+
) or both (A27L
−
D8L
−
). The A27L
+
D8L
+
and A27L
−
D8L
+
mutants grew well in BSC40 cells, consistent with previous observations. However, the IMV titers of A27L
+
D8L
−
and A27L
−
D8L
−
viruses in BSC40 cells were reduced, reaching only 10% of the level for the control virus. The data suggested an important role for D8L protein in WR32-7/Ind14K virus growth in cell cultures. A27L protein, on the other hand, could not complement the functions of D8L protein. The low titers of the A27L
+
D8L
−
and A27L
−
D8L
−
mutant viruses were not due to defects in the morphogenesis of IMV, and the mutant virions demonstrated a brick shape similar to that of the control virions. Furthermore, the infectivities of the A27L
+
D8L
−
and A27L
−
D8L
−
mutant virions were 6 to 10% of that of the A27L
+
D8L
+
control virus. Virion binding assays revealed that A27L
+
D8L
−
and A27L
−
D8L
−
mutant virions bound less well to BSC40 cells, indicating that binding of viral D8L protein to cell surface chondroitin sulfate could be important for vaccinia virus entry.
Possible Interactions between the NS-1 Protein and Tumor Necrosis Factor Alpha Pathways in Erythroid Cell Apoptosis Induced by Human Parvovirus B19
Authors: Sol N. , Le Junter J. , Vassias I. , Freyssinier J. M. , Thomas A. , Prigent A. F. , Rudkin B. B. , Fichelson S. , Morinet F.
Published: December 2019

Human erythroid progenitor cells are the main target cells of the human parvovirus B19 (B19), and B19 infection induces a transient erythroid aplastic crisis. Several authors have reported that the nonstructural protein 1 (NS-1) encoded by this virus has a cytotoxic effect, but the underlying mechanism of NS-1-induced primary erythroid cell death is still not clear. In human erythroid progenitor cells, we investigated the molecular mechanisms leading to apoptosis after natural infection of these cells by the B19 virus. The cytotoxicity of NS-1 was concomitantly evaluated in transfected erythroid cells. B19 infection and NS-1 expression induced DNA fragmentation characteristic of apoptosis, and the commitment of erythroid cells to undergo apoptosis was combined with their accumulation in the G
2
phase of the cell cycle. Since B19- and NS-1-induced apoptosis was inhibited by caspase 3, 6, and 8 inhibitors, and substantial caspase 3, 6, and 8 activities were induced by NS-1 expression, there may have been interactions between NS-1 and the apoptotic pathways of the death receptors tumor necrosis factor receptor 1 and Fas. Our results suggest that Fas-FasL interaction was not involved in NS-1- or B19-induced apoptosis in erythroid cells. In contrast, these cells were sensitized to tumor necrosis factor alpha (TNF-α)-induced apoptosis. Moreover, the ceramide level was enhanced by B19 infection and NS-1 expression. Therefore, our results suggest that there may be a connection between the respective apoptotic pathways activated by TNF-α and NS-1 in human erythroid cells.
Evidence that the Genomic RNA of Hepatitis E Virus Is Capped
Authors: Kabrane-Lazizi Yamina , Meng Xiang-Jin , Purcell Robert H. , Emerson Suzanne U.
Published: December 2019

Hepatitis E virus (HEV) is an unclassified virus with a positive-sense RNA genome and an undefined replication strategy. In order to determine whether the HEV genome is capped or not, we developed a reverse transcription-PCR assay that is based on the ability of a monoclonal antibody to recognize 7-methylguanosine (m
7
G). Antibody to m
7
G bound RNA extracted from virions of two different HEV genotypes. The cap analog competitively inhibited the binding of virion RNAs, demonstrating that HEV has a capped RNA genome.
Herpes Simplex Virus Inhibits Apoptosis through the Action of Two Genes, Us5 and Us3
Authors: Jerome Keith R. , Fox Richard , Chen Zheng , Sears Amy E. , Lee Hyung-yul , Corey Lawrence
Published: December 2019

Apoptosis of virus-infected cells occurs either as a direct response to viral infection or upon recognition of infection by the host immune response. Apoptosis reduces production of new virus from these cells, and therefore viruses have evolved inhibitory mechanisms. We previously showed that laboratory strains of herpes simplex virus type 1 (HSV-1) protect infected cells from apoptosis induced by cytotoxic T lymphocytes or ethanol. We have now evaluated the ability of HSV-1 and HSV-2 laboratory and clinical isolates to inhibit apoptosis induced by anti-Fas antibody or UV irradiation and explored the genetic basis for this inhibition. HSV-1 isolates inhibited apoptosis induced by UV or anti-Fas antibody. In contrast, HSV-2 clinical isolates failed to inhibit apoptosis induced by either stimulus, although the HSV-2 laboratory strain 333 had a partial inhibitory effect on UV-induced apoptosis. Inhibition of apoptosis by HSV was accompanied by marked reduction of caspase-3 and caspase-8 activity. Deletion of the HSV-1 Us3 gene markedly reduced inhibition of UV-induced apoptosis and partially abrogated inhibition of Fas-mediated apoptosis. Conversely, deletion of the HSV-1 Us5 gene markedly reduced protection from Fas-mediated apoptosis and partially abrogated protection from UV. The Us11 and Us12 genes were not necessary for protection from apoptosis induced by either stimulus. The differences between HSV-1 and HSV-2 in the ability to inhibit apoptosis may be factors in the immunobiology of HSV infections.
Palmitoylation of the Intracytoplasmic R Peptide of the Transmembrane Envelope Protein in Moloney Murine Leukemia Virus
Authors: Olsen Katharina E. P. , Andersen Klaus B.
Published: December 2019

Previously it was reported that the 16-amino-acid (aa) C-terminal cytoplasmic tail of Moloney murine leukemia virus (MoMLV) transmembrane protein Pr15E is cleaved off during virus synthesis, yielding the mature, fusion active transmembrane protein p15E and the 16-aa peptide (R peptide or p2E). It remains to be elucidated how the R peptide impairs fusion activity of the uncleaved Pr15E. The R peptide from MoMLV was analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostained with antiserum against the synthetic 16-aa R peptide. The R peptide resolved with an apparent molecular mass of 7 kDa and not the 4 kDa seen with the corresponding synthetic peptide. The 7-kDa R peptide was found to be membrane bound in MoMLV-infected NIH 3T3 cells, showing that cleavage of the 7-kDa R-peptide tail must occur before or during budding of progeny virions, in which only small amounts of the 7-kDa R peptide were found. The 7-kDa R peptide was palmitoylated since it could be labeled with [
3
H]palmitic acid, which explains its membrane association, slower migration on gels, and high sensitivity in immunoblotting. The present results are in contrast to previous findings showing equimolar amounts of R peptide and p15E in virions. The discrepancy, however, can be explained by the presence of nonpalmitoylated R peptide in virions, which were poorly detected by immunoblotting. A mechanistic model is proposed. The uncleaved R peptide can, due to its lipid modification, control the conformation of the ectodomain of the transmembrane protein and thereby govern membrane fusion.
Structure-Based Mutagenesis of the Human Immunodeficiency Virus Type 1 DNA Attachment Site: Effects on Integration and cDNA Synthesis
Authors: Brown Heidi E. V. , Chen Hongmin , Engelman Alan
Published: December 2019

Sequences at the ends of linear retroviral cDNA important for integration define the viral DNA attachment (att) site. Whereas determinants of human immunodeficiency virus type 1 (HIV-1) integrase important for replication in T lymphocytes have been extensively characterized, regions of theattsite important for viral spread have not been thoroughly examined. Previous transposon-mediated footprinting of preintegration complexes isolated from infected cells revealed enhanced regions of bacteriophage Mu insertion near the ends of HIV-1 cDNA, in the regions of theattsites. Here, we identified the subterminal cDNA sequences cleaved during in vitro footprinting and used this structure-based information together with results of previous work to construct and characterize 24attsite mutant viruses. We found that although subterminal cDNA sequences contributed to HIV-1 replication, the identities of these bases were not critical for integration. In contrast, the phylogenetically conserved CA dinucleotides located at the ends of HIV-1 contributed significantly to virus replication and integration. Mutants containing one intact CA end displayed delays in peak virus growth compared to the wild type. In contrast, double mutant viruses lacking both CAs were replication defective. The A of the CA appeared to be the most critical determinant of integration, because two different U5 mutant viruses containing the substitution of TG for CA partially reverted by changing the G back to A. We also identified a U5 deletion mutant in which the CA played a crucial role in reverse transcription.
Genetic Dissection of Cell Growth Arrest Functions Mediated by the Epstein-Barr Virus Lytic Gene Product, Zta
Authors: Rodriguez Antonio , Armstrong Monica , Dwyer Daniel , Flemington Erik
Published: December 2019

Expression of the Epstein-Barr virus (EBV) latency-associated genes activates cell cycle progression and drives immortalization of the infected cell. In contrast, progression of the EBV replication program occurs most efficiently in growth-arrested cells. Previous studies showed that the EBV-encoded immediate-early transcription factor, Zta, can induce expression of the cyclin-dependent kinase inhibitors, p21 and p27, the tumor suppressor, p53, and cell growth arrest. Moreover, Zta-mediated induction of growth arrest occurs independently of its transcriptional transactivation function. Here we show that substitution of Zta’s basic DNA binding domain with the analogous region of the Zta homologue, c-Fos, abrogates Zta’s ability to induce growth arrest and to induce p21, p27, or p53 expression, suggesting that protein-protein interactions between this region of Zta and key cell cycle control proteins are involved in signaling cell cycle arrest. We also show that despite the crucial role for Zta’s basic domain in eliciting cell growth arrest, its amino terminus is required for efficient induction of p27 and it modulates the level of p53 induction. Last, we provide evidence that Zta-mediated inductions of p21, p27, and p53 occur, at least in part, through distinct pathways. Therefore, Zta interacts with multiple growth arrest pathways, a property which may have evolved partly as a means to ensure that lytic replication occurs in a growth-arrested setting in multiple different tissues in various states of differentiation.
Phosphorylation of Simian Cytomegalovirus Assembly Protein Precursor (pAPNG.5) and Proteinase Precursor (pAPNG1): Multiple Attachment Sites Identified, Including Two Adjacent Serines in a Casein Kinase II Consensus Sequence
Authors: Plafker Scott M. , Woods Amina S. , Gibson Wade
Published: December 2019

The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homologs in other herpesviruses, functions at several key steps during the process of capsid formation. This protein, and the genetically related maturational proteinase, is distinguished from the other capsid proteins by posttranslational modifications, including phosphorylation. The objective of this study was to identify sites at which pAP is phosphorylated so that the functional significance of this modification and the enzyme(s) responsible for it can be determined. In the work reported here, we used peptide mapping, mass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated. The other site(s) is in a different domain of the protein, is phosphorylated less frequently than the CKII site, does not require preceding CKII-site phosphorylation, and causes an electrophoretic mobility shift when phosphorylated. Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the proteinase or on the substrate behavior of pAP. Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications can be made by a cellular enzyme(s). Apparent compartmental differences in phosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modifications.
Identification of Functional Domains in the 14-Kilodalton Envelope Protein (A27L) of Vaccinia Virus
Authors: Vázquez María-Isabel , Esteban Mariano
Published: December 2019

The mechanism of entry of vaccinia virus (VV) into cells is still a poorly understood process. A 14-kDa protein (encoded by the A27L gene) in the envelope of intracellular mature virus (IMV) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells. We have previously described the structural organization of the VV 14-kDa protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted Leu zipper-like third alpha helix with an important role in the interaction with a 21-kDa membrane protein (encoded by the A17L gene) thought to anchor the 14-kDa protein to the envelope of IMV (M.-I. Vázquez, G. Rivas, D. Cregut, L. Serrano, and M. Esteban, J. Virol. 72:10126–10137, 1998). To identify the functional domains important for virus entry and release, we have generated VV recombinants containing a copy of the A27L gene regulated by the
lacI
operator-repressor system of
Escherichia coli
(VVIndA27L) in the thymidine kinase locus and a mutant form of the A27L gene in the hemagglutinin locus but expressed constitutively under the control of an early-late VV promoter. Cells infected with a VV recombinant that expresses a mutant 14-kDa form lacking the first 29 amino acids at the N terminus failed to form extracellular enveloped virus (EEV). Fusion-from-without assays with purified virus confirmed that the fusion process was mediated by the 14-kDa protein and the fusion domain to be contained within amino acids 29 to 43 of the N-terminal region. Competitive inhibition of the infection process with soluble heparin and synthetic peptides and in vitro experiments with purified mutant proteins identified the heparin binding domain within amino acids 21 to 33, suggesting that this domain is involved in virus-cell binding via heparan sulfate. Thus, the N terminus of the 14-kDa protein contains a heparin binding domain, a fusion domain, and a domain responsible for interacting with proteins or lipids in the Golgi stacks for EEV formation and virus spread.
Hierarchal Utilization of Different T-Cell Receptor Vβ Gene Segments in the CD8+-T-Cell Response to an Immunodominant Moloney Leukemia Virus-Encoded Epitope In Vivo
Authors: Brawand Pierre , Cerottini Jean-Charles , MacDonald H. Robson
Published: December 2019

The CD8+-T-cell response to Moloney murine leukemia virus (M-MuLV)-associated antigens in C57BL/6 mice is directed against an immunodominantgag-encoded epitope (CCLCLTVFL) presented in the context ofH-2Dband is restricted primarily to cytotoxic T lymphocytes (CTL) expressing the Vα3.2 and Vβ5.2 gene segments. We decided to examine the M-MuLV response in congenic C57BL/6 Vβamice which are unable to express the dominant Vα3.2+Vβ5.2+T-cell receptor (TCR) due to a large deletion at the TCR locus that includes the Vβ5.2 gene segment. Interestingly, M-MuLV-immune C57BL/6 Vβamice were still able to reject M-MuLV-infected tumor cells and direct ex vivo analysis of peripheral blood lymphocytes from these immune mice revealed a dramatic increase in CD8+cells utilizing the same Vα3.2 gene segment in association with two different Vβ segments (Vβ3 and Vβ17). Surprisingly, all these CTL recognized the same immunodominant M-MuLVgagepitope. Analysis of the TCR repertoire of individual M-MuLV-immune (C57BL/6 × C57BL/6 Vβa)F1mice revealed a clear hierarchy in Vβ utilization, with a preferential usage of the Vβ17 gene segment, whereas Vβ3 and especially Vβ5.2 were used to much lesser extents. Sequencing of TCRα- and -β-chain junctional regions of CTL clones specific for the M-MuLVgagepitope revealed a diverse repertoire of TCRβ chains in Vβamice and a highly restricted TCRβ-chain repertoire in Vβbmice, whereas TCRα-chain sequences were highly conserved in both cases. Collectively, our data indicate that theH-2Db-restricted M-MuLVgagepitope can be recognized in a hierarchal fashion by different Vβ domains and that the degree of β-chain diversity varies according to Vβ utilization.
Sequence and Insertion Sites of Murine Melanoma-Associated Retrovirus
Authors: Li Mengfeng , Huang Xiaojun , Zhu Zhenyu , Gorelik Elieser
Published: December 2019

We previously showed that B16 melanoma cells produce ecotropic melanoma-associated retrovirus (MelARV) which encodes a melanoma-associated antigen recognized by MM2-9B6 monoclonal antibody. The biological significance of MelARV in melanoma formation remains unknown. We found that infection of normal melanocytes with MelARV resulted in malignant transformation. It is likely that MelARV emerged from the defective Emv-2 provirus, a single copy of ecotropic provirus existing in the genome of C57BL/6 mice. In the present study, we cloned and sequenced the full-length MelARV genome and its insertion sites and we completed sequencing of the Emv-2 provirus. Our data show that MelARV has a typical full-length retroviral genome with high homology (98.54%) to Emv-2, indicating a close relationship between both viruses. MelARV probably emerged as a result of recombination between Emv-2 and an endogenous nonecotropic provirus. Some observed differences in the
gag
and
pol
regions of MelARV might account for the restoration of productivity and infectivity of a novel retrovirus that somatically emerged during melanoma formation. MelARV does not contain any oncogene and therefore might induce transformation by insertional mutagenesis. We sequenced two insertion sites of MelARV. The first insertion site represents the 3′ coding region of the c-
maf
proto-oncogene at 67.0 centimorgans (cM) on chromosome 8. The c-
maf
proto-oncogene encodes a basic leucine zipper protein homologous to c-
fos
and c-
jun
. Insertion of MelARV in BL6 melanoma cells resulted in the up-regulation of c-
maf
. It is noteworthy that the Emv-2 provirus is also inserted into a noncoding region at 61.0 cM on the same chromosome 8. The second insertion site is the 3′ noncoding region of the DNA polymerase gamma (PolG) gene on chromosome 7. The expression of PolG was not affected by the MelARV insertion. Further investigation of the biological significance of MelARV in melanoma formation is being undertaken.