bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of caprine arthritis-encephalitis virus long terminal repeat by gamma interferon
Authors: Tong-Starksen S E , Sepp T , Pagtakhan A S
Published: January 2020

Caprine arthritis-encephalitis virus (CAEV) is a lymphotropic lentivirus whose replication increases during monocyte maturation. We examined gene expression directed by the CAEV long terminal repeat (LTR) in a promonocytic cell line stimulated with several agents. Our results demonstrate that the CAEV LTR is activated by treatment of immature monocytes with gamma interferon (IFN-gamma) or a phorbol ester but not with tumor necrosis factor alpha or lipopolysaccharide. The cis-acting element in the LTR for the IFN-gamma response localizes to a duplicated 70-bp motif that contains an IFN-gamma response element, the gamma-activated site. One copy of the motif is necessary and sufficient for the response to IFN-gamma. Multiple copies contribute to basal transcriptional activity in the context of a heterologous promoter. This IFN-gamma response element in the CAEV LTR differs from the element required for the response to phorbol esters. Thus, activation of the CAEV LTR in monocytes that are stimulated by IFN-gamma, a cytokine that is secreted in response to viral infections, could contribute to conversion from latent to high-level viral replication in infected hosts.
Sequence variability of Borna disease virus open reading frame II found in human peripheral blood mononuclear cells
Authors: Kishi M , Arimura Y , Ikuta K , Shoya Y , Lai P K , Kakinuma M
Published: January 2020

A cDNA fragment of the Borna disease virus (BDV) open reading frame II (ORF-II), which encodes a 24-kDa phosphoprotein (p24 [P protein]), was amplified from total RNA of peripheral blood mononuclear cells (PBMC) from three psychiatric inpatients. The amplified cDNA fragments were cloned, sequenced, and analyzed. A total of 15 clones, 5 from each patient, were studied. Intrapatient divergencies of the BDV ORF-II nucleotide sequence were 4.2 to 7.3%, 4.8 to 7.3%, and 2.8 to 7.1% for the three patients, leading to differences of 7.7 to 14.5%, 10.3 to 17.1%, and 6.0 to 16.2%, respectively, in the deduced amino acid sequence for BDV p24. Interpatient divergencies among the 15 clones were 5.9 to 12.7% at the nucleotide level and 12.8 to 28.2% at the amino acid level. Thus, in p24, BDV in human PBMC of the patients undergoes mutation at high rates in vivo. Additionally, we found that the nucleotide sequence of the 15 human BDV ORF-II cDNA clones differed from those of the horse strains V and He/80-1 by 4.2 to 9.3%. However, comparison of the consensus amino acid sequence deduced from the 15 human clones with those of the horse strains revealed no human-specific amino acid residue, suggesting that the BDV infecting humans may be related to that infecting horses.
Hepatitis B virus pX activates NF-kappa B-dependent transcription through a Raf-independent pathway
Authors: Chirillo P , Falco M , Puri P L , Artini M , Balsano C , Levrero M , Natoli G
Published: January 2020

In this study, we characterized the molecular events involved in the activation of the ubiquitous transcription factor NF-kappa B by the viral transactivator pX. pX expression in HeLa cells determines a manyfold increase in NF-kappa B-dependent transcription, which is associated with an increase in p50/p65 heterodimer DNA-binding activity. Since the I kappa B-alpha inhibitory subunit proteolytic degradation, which follows its phosphorylation/modification, is a key event in NF-kappa B activation by different stimuli (such as growth factors, phorbol esters, tumor necrosis factor, UV irradiation, and oxygen radicals), we investigated pX effects on I kappa B-alpha, as well as the possible involvement of known signalling pathways in pX-induced NF-kappa B-dependent transcription. We observed that although pX had no direct effect on p50 or p65, it was able to restore the I kappa B-alpha-suppressed p50/p65 activity. More directly, the stable expression of pX in HeLa cells resulted in reduced levels of I kappa B-alpha in the cytoplasm. Pretreatment of the cells with H7, calphostin C, tyrphostin 25, or N-acetylcysteine did not impair the effects of pX on NF-kappa B, thus ruling out the involvement of protein kinase C, tyrosine kinases, and oxygen radicals. Finally, while most of the known NF-kappa B-activating agents converge on Raf-1 protein kinase, when Raf-1 activity is blocked by overexpression of a dominant negative mutant, the effects of pX on NF-kappa B are not impaired. Thus, we suggest that although pX is able to activate the Ras/Raf-1-signalling pathway, it triggers NF-kappa B activation by an as yet unidentified Raf-1-independent pathway.
bcl-2 alters influenza virus yield, spread, and hemagglutinin glycosylation
Authors: Olsen C W , Kehren J C , Dybdahl-Sissoko N R , Hinshaw V S
Published: January 2020

We previously demonstrated that expression of bcl-2 in Madin-Darby canine kidney (MDCK) cells blocks influenza virus-induced apoptosis and DNA fragmentation. We show here that expression of bcl-2 also reduces the level of infectious virus production and the spread of virus in MDCK cell cultures infected at a low multiplicity of infection. This effect is associated with modified glycosylation of the hemagglutinin protein.
Specific binding of human immunodeficiency virus type 1 (HIV-1) Gag-derived proteins to a 5' HIV-1 genomic RNA sequence
Authors: Geigenmüller U , Linial M L
Published: January 2020

We developed an in vitro binding assay to study the specific interaction between human immunodeficiency virus type 1 (HIV-1) RNA and the Gag polyprotein. Binding of the in vitro-expressed protein to in vitro-transcribed RNA was determined by altered migration of the protein in polyacrylamide gels. We found that a Gag precursor lacking the matrix domain bound specifically to HIV-1 RNA, while deletion of both matrix and capsid domains diminished the specificity of binding. Among several regions of HIV-1 RNA tested, strongest binding was seen with the 5'-most 261 nucleotides, while antisense RNA from the same region did not bind.
Multiple specificities in the murine CD4+ and CD8+ T-cell response to dengue virus
Authors: Rothman A L , Kurane I , Ennis F A
Published: January 2020

The target epitopes, serotype specificity, and cytolytic function of dengue virus-specific T cells may influence their theoretical roles in protection against secondary infection as well as the immunopathogenesis of dengue hemorrhagic fever. To study these factors in an experimental system, we isolated dengue virus-specific CD4+ and CD8+ T-cell clones from dengue-2 virus-immunized BALB/c mice. The T-cell response to dengue virus in this mouse strain was heterogeneous; we identified at least five different CD4+ phenotypes and six different CD8+ phenotypes. Individual T-cell clones recognized epitopes on the dengue virus pre-M, E, NSl/NS2A, and NS3 proteins and were restricted by the I-Ad, I-Ed, Ld, and Kd antigens. Both serotype-specific and serotype-cross-reactive clones were isolated in the CD4+ and CD8+ subsets; among CD8+ clones, those that recognized the dengue virus structural proteins were serotype specific whereas those that recognized the nonstructural proteins were serotype cross-reactive. All of the CD8+ and one of five CD4+ clones lysed dengue virus-infected target cells. Using synthetic peptides, we identified an Ld-restricted epitope on the E protein (residues 331 to 339, SPCKIPFEI) and a Kd-restricted epitope on the NS3 protein (residues 296 to 310, ARGYISTRVEM GEAA). These data parallel previous findings of studies using human dengue virus-specific T-cell clones. This experimental mouse system may be useful for studying the role of the virus serotype and HLA haplotype on T-cell responses after primary dengue virus infection.
Attachment and entry of recombinant Norwalk virus capsids to cultured human and animal cell lines
Authors: White L J , Ball J M , Hardy M E , Tanaka T N , Kitamoto N , Estes M K
Published: January 2020

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region of the capsid protein is involved in specific binding of rNV VLPs to cells.
Evolution of a persistent aphthovirus in cytolytic infections: partial reversion of phenotypic traits accompanied by genetic diversification
Authors: Sevilla N , Domingo E
Published: January 2020

Foot-and-mouth disease virus (FMDV) shows a dual potential to be cytolytic or to establish persistent infections in cell culture. FMDV R100, a virus rescued after 100 passages of carrier BHK-21 cells persistently infected with FMDV clone C-S8c1, showed multiple genetic and phenotypic alterations relative to the parental clone C-S8c1. Several FMDV R100 populations have been subjected to 100 serial cytolytic infections in BHK-21 cells, and the reversion of phenotypic and genetic alterations has been analyzed. An extreme temperature sensitivity of R100 reverted totally or partially in some passage series but not in others. The small-plaque morphology reverted to normal size in all cases. The hypervirulence for BHK-21 cells did not revert, and even showed an increase, upon cytolytic passage. Most of the mutations that had been fixed in the R100 genome during persistence did not revert in the course of cytolytic passages, but the extended polyribocytidylate tract of R100 (about 460 residues, versus 290 in C-S8c1) decreased dramatically in length, to the range of 220 to 260 residues in all passage series examined. In passages involving very large viral populations, a variant with two amino acid substitutions (L-144-->V and A-145-->P) next to the highly conserved Arg-Gly-Asp (RGD motif; positions 141 to 143) within the G-H loop of capsid protein VP1 became dominant. A clonal analysis allowed isolation of a mutant with the single replacement A-145-->P. Viral production and growth competition experiments showed the two variants to have a fitness very close to that of the parental virus. The results provide evidence that the repertoire of variants that could potentially become dominant in viral quasispecies may be influenced by the population size of the evolving virus. The net results of a series of persistent-infection passages followed by a series of cytolytic passages was progressive genomic diversification despite reversion or stasis of phenotypic traits. Implications for the evolution of RNA viruses are discussed.
Influenza virus hemagglutinin and neuraminidase glycoproteins stimulate the membrane association of the matrix protein
Authors: Enami M , Enami K
Published: January 2020

We have analyzed the mechanism by which the matrix (M1) protein associates with cellular membranes during influenza A virus assembly. Interaction of the M1 protein with the viral hemagglutinin (HA) or neuraminidase (NA) glycoprotein was extensively analyzed by using wild-type and transfectant influenza viruses as well as recombinant vaccinia viruses expressing the M1 protein, HA, or NA. Membrane binding of the M1 protein was significantly stimulated at the late stage of virus infection. Using recombinant vaccinia viruses, we found that a relatively small fraction (20 to 40%) of the cytoplasmic M1 protein associated with cellular membranes in the absence of other viral proteins, while coexpression of the HA and the NA stimulated membrane binding of the M1 protein. The stimulatory effect of the NA (>90%) was significant and higher than that of the HA (>60%). Introduction of mutations into the cytoplasmic tail of the NA interfered with its stimulatory effect. Meanwhile, the HA may complement the defective NA and facilitate virus assembly in cells infected with the NA/TAIL(-) transfectant. In conclusion, the highly conserved cytoplasmic tails of the HA and NA play an important role in virus assembly.
Transgenic mice expressing human measles virus (MV) receptor CD46 provide cells exhibiting different permissivities to MV infections
Authors: Horvat B , Rivailler P , Varior-Krishnan G , Cardoso A , Gerlier D , Rabourdin-Combe C
Published: January 2020

We have generated transgenic mice ubiquitously expressing the human receptor for measles virus (MV), CD46 (membrane cofactor protein). Various cell types were isolated from these transgenic mice and analyzed for their ability to support MV replication in vitro. Although MV could enter into all CD46-expressing cells, differential susceptibilities to MV infection were detected depending on the cell type. Cell cultures obtained from transgenic lungs and kidneys were found to be permissive of MV infection, since RNA specific for MV genes was detected and viral particles were released, although at a low level. Similarly to human lymphocytes, activated T and B lymphocytes isolated from transgenic mice could support MV replication; virus could enter, transcribe viral RNA, and produce new infectious particles. When expressing viral proteins, lymphocytes down-regulated CD46 from the surface. Interestingly, while activated T lymphocytes from nontransgenic mice did not support MV infection, activated nontransgenic murine B lymphocytes replicated MV as well as transgenic B lymphocytes, suggesting the use of an alternative virus receptor for entry. In contrast to the previous cell types, murine peritoneal and bone marrow-derived macrophages, regardless of whether they were activated, could not support MV replication. Furthermore, although MV entered into macrophages and virus-specific RNA transcription occurred, no virus protein or infectious virus particles could be detected. These results show the importance of the particular cell-type-specific host factors for MV replication in murine cells which may be responsible for the differential permissivity of MV infection.
Reovirus-induced acute myocarditis in mice correlates with viral RNA synthesis rather than generation of infectious virus in cardiac myocytes
Authors: Sherry B , Baty C J , Blum M A
Published: January 2020

The capacity for different reovirus reassortant viruses to induce acute myocarditis in mice correlates with cytopathogenic effect in primary cultures of murine cardiac myocytes. Multiple viral genes encoding proteins involved in viral RNA synthesis are determinants of this disease. We therefore evaluated the role of viral RNA synthesis in induction of acute myocarditis by infecting primary cultures of cardiac myocytes with a panel of myocarditic and nonmyocarditic viruses and quantitating RNA synthesis. RNA synthesis correlated with induction of myocarditis and with the S1 and M1 reovirus genes. Since one consequence of viral RNA synthesis is generation of infectious virus, we looked next at viral yield from cardiac myocyte cultures. Yield of infectious virus at an early time postinfection or as a final yield from primary infections did not correlate with myocarditis, but instead both correlated with the S1 gene. The S1 gene also determined the fraction of cells infected during primary infections in the culture, which varied dramatically between viruses. Viral yields per infected cell were similar for most myocarditic and nonmyocarditic reoviruses and did not correlate with induction of myocarditis or any reovirus gene. Together, the data provide two insights into reovirus-induced acute myocarditis in mice. First, while the S1 gene. which encodes the viral attachment protein sigma1 (as well as a nonstructural protein, sigma1s, of unknown function) does not determine the myocarditic potential of these viruses, it does determine the efficiency with which they infect cardiac myocytes. Second, while viral RNA synthesis is a determinant of acute myocarditis, this is not due to generation of infectious virus. This finding suggests that some other consequence of viral RNA synthesis, for example, induction of interferon, may determine reovirus-induced acute myocarditis.
Influenza virus polymerase basic protein 1 interacts with influenza virus polymerase basic protein 2 at multiple sites
Authors: Biswas S K , Nayak D P
Published: January 2020

Three polymerase proteins of influenza type A virus interact with each other to form the active polymerase complex. Polymerase basic protein 1 (PB1) can interact with PB2 in the presence or absence of polymerase acidic protein. In this study, we investigated the domains of PB1 involved in complex formation with PB2 in vivo, using coexpression and coimmunoprecipitation of the PB1-PB2 complex with monospecific antibodies. Results show that PB1 possesses at least two regions which can interact independently and form stable complexes with PB2. Both of these regions are located at the NH2 terminus of PB1; the COOH-terminal half of PB1 is not involved in interacting with PB2. Deletion analysis further demonstrated that the interacting regions of PB1 encompass amino acids (aa) 48 to 145 and aa 251 to 321. Linker insertions throughout the PB1 sequences did not affect complex formation with PB2. Deletion and linker-insertion mutants of PB1 were tested for polymerase activity in vivo. For this analysis, we developed a simplified assay for viral polymerase activity that uses a reporter chloramphenicol acetyltransferase gene containing the 5' and 3' ends of influenza viral promoter and nontranslating regions (minus sense) of the NS gene joined to a hepatitis delta virus ribozyme at its 3' end. This assay demonstrated that all deletion mutants of PB1 exhibited either background or greatly reduced polymerase activity irrespective of the ability to interact with PB2 and that all linker-insertion mutants except one at the extreme COOH end (L-746) of PB1 were also negative for viral polymerase activity. These results show that compared with complex formation of PB1 with PB2, the polymerase activity of PB1 was extremely sensitive to structural perturbation.
Role of the DIS hairpin in replication of human immunodeficiency virus type 1
Authors: Berkhout B , van Wamel J L
Published: January 2020

The virion-associated genome of human immunodeficiency virus type 1 consists of a noncovalently linked dimer of two identical, unspliced RNA molecules. A hairpin structure within the untranslated leader transcript is postulated to play a role in RNA dimerization through base pairing of the autocomplementary loop sequences. This hairpin motif with the palindromic loop sequence is referred to as the dimer initiation site (DIS), and the type of interaction is termed loop-loop kissing. Detailed phylogenetic analysis of the DIS motifs in different human and simian immunodeficiency viruses revealed conservation of the hairpin structure with a 6-mer palindrome in the loop, despite considerable sequence divergence. This finding supports the loop-loop kissing mechanism. To test this possibility, proviral genomes with mutations in the DIS palindrome were constructed. The appearance of infectious virus upon transfection into SupT1 T cells was delayed for the DIS mutants compared with that obtained by transfection of the wild-type provirus (pLAI), confirming that this RNA motif plays an important role in virus replication. Surprisingly, the RNA genome extracted from mutant virions was found to be fully dimeric and to have a normal thermal stability. These results indicate that the DIS motif is not essential for human immunodeficiency virus type 1 RNA dimerization and suggest that DIS base pairing does not contribute to the stability of the mature RNA dimer. Instead, we measured a reduction in the amount of viral RNA encapsidated in the mutant virions, suggesting a role of the DIS motif in RNA packaging. This result correlates with the idea that the processes of RNA dimerization and packaging are intrinsically linked, and we propose that DIS pairing is a prerequisite for RNA packaging.
Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68
Authors: Weck K E , Barkon M L , Yoo L I , Speck S H , Virgin HW I V
Published: January 2020

Murine gammaherpesvirus 68 (gamma HV-68; also referred to as MHV-68) is a gammaherpesvirus which infects murid rodents. Previous studies showed that CD8 T cells are important for controlling gamma HV-68 replication during the first 2 weeks of infection and suggested a role for B cells in latent or persistent gamma HV-68 infection. To further define the importance of B cells and CD8 T cells during acute and chronic gamma HV-68 infection, we examined splenic infection in mice with null mutations in the transmembrane domain of the mu-heavy-chain constant region (MuMT; B-cell and antibody deficient) or in the beta2-microglobulin gene (beta2 -/-; CD8 deficient). Immunocompetent mice infected intraperitoneally with gamma HV-68 demonstrated peak splenic titers 9 to 10 days postinfection, cleared infectious virus 15 to 20 days postinfection, and harbored low levels of latent virus at 6 weeks postinfection. Beta2-/- mice showed peak splenic gamma HV-68 titers similar to those of normal mice but were unable to clear infectious virus completely from the spleen, demonstrating persistent infectious virus 6 weeks postinfection. These data indicate that CD8 T cells are important for clearing infectious gamma HV-68 from the spleen. Infected MuMT mice did not demonstrate detectable infectious gamma HV-68 in the spleen at any time after infection, indicating that mature B lymphocytes are necessary for acute splenic infection by gamma HV-68. Despite the lack of measurable acute infection, MuMT spleen cells harbored latent virus 6 weeks postinfection at a level about 100-fold higher than that in normal mice. These data demonstrate establishment of latency by a herpesvirus in an organ in the absence of acute viral replication in that organ. In addition, they demonstrate that gamma HV-68 can establish latency in a cell type other than mature B lymphocytes.
Study of mother-to-child Epstein-Barr virus transmission by means of nested PCRs
Authors: Meyohas M C , Maréchal V , Desire N , Bouillie J , Frottier J , Nicolas J C
Published: January 2020

The aim of this study was to investigate the possibility of vertical Epstein-Barr virus (EBV) transmission. We developed two nested-PCR methods for amplifying distinct regions of EBV DNA (BNRF1 and BamHI W) in circulating lymphocytes. Nested PCR was applied to samples obtained from 67 mother-infant pairs within 1 week of birth. We also tested samples from 16 neonates born to human immunodeficiency virus (HIV)-infected mothers to determine whether HIV increases the potential risk of vertical EBV transmission. About half of the 67 women in the first population were positive by nested PCR. Two neonates born to EBV PCR-positive women were also PCR positive. One of the 16 neonates born to HIV-infected women was PCR positive for EBV. These results strongly support the possibility of EBV transmission in utero or during delivery but do not suggest that HIV infection increases this risk. Further studies are required to confirm these findings, to identify the precise mode of vertical EBV transmission, and to determine the outcome for infants who are positive at birth for EBV DNA by nested PCR.
Lack of integrase can markedly affect human immunodeficiency virus type 1 particle production in the presence of an active viral protease
Authors: Bukovsky A , Göttlinger H
Published: January 2020

The Gag-Pol polyprotein of human immunodeficiency virus type 1 is not required for efficient viral particle assembly or release. However, in this report we demonstrate that the synthesis of a truncated Gag-Pol precursor due to a premature termination codon in pol can reduce the ability of a full-length provirus to direct the formation of viral particles. Marked effects on particle production were seen when premature termination codons were introduced into the integrase (IN)-coding region. By contrast, a mutant which lacked both IN and reverse transcriptase (RT) formed particles with normal efficiency. Particle production by IN mutants was restored to wild-type levels when a second premature termination codon was introduced at the 5' end of the RT-coding sequence. Particle formation was similarly restored by a second site mutation in the viral protease (PR) gene which prevented proteolytic processing of the Gag polyprotein. Finally particle formation was restored in the presence of A77003, a specific inhibitor of human immunodeficiency virus type 1 PR. These results suggest that the effects of a lack of IN sequences on particle formation require the synthesis of a Gag-Pol precursor which contains RT sequences and are due to inappropriate PR activity.
Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation
Authors: Demers G W , Espling E , Harry J B , Etscheid B G , Galloway D A
Published: January 2020

Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals.
The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice
Authors: Guidotti L G , Matzke B , Pasquinelli C , Shoenberger J M , Rogler C E , Chisari F V
Published: January 2020

In this study, we examined the ability of the hepatitis B virus (HBV) precore, envelope, and X gene products to modulate HBV replication in the livers of transgenic mice that replicate the virus. Hepatic HBV replication was not affected by overexpression of the envelope or X gene products when these animals were crossed with transgenic mice that express the corresponding viral genes in the hepatocyte. Overexpression of the precore protein, however, eliminated nucleocapsid particles from the cytoplasm of the hepatocytes and abolished HBV replication without affecting the hepatic steady-state content of pregenomic HBV RNA. These observations suggest that the precore protein can exert a dominant negative effect on HBV replication, presumably at the level of nucleocapsid particle maturation or stability, suggesting an important role for this enigmatic viral protein in the HBV life cycle.
The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels
Authors: Ewart G D , Sutherland T , Gage P W , Cox G B
Published: January 2020

Vpu is a small phosphorylated integral membrane protein encoded by the human immunodeficiency virus type 1 genome and found in the endoplasmic reticulum and Golgi membranes of infected cells. It has been linked to roles in virus particle budding and degradation of CD4 in the endoplasmic reticulum. However, the molecular mechanisms employed by Vpu in performance of these functions are unknown. Structural similarities between Vpu and the M2 protein of influenza A virus have raised the question of whether the two proteins are functionally analogous: M2 has been demonstrated to form cation-selective ion channels in phospholipid membranes. In this paper we provide evidence that Vpu, purified after expression in Escherichia coli, also forms ion channels in planar lipid bilayers. The channels are approximately five- to sixfold more permeable to sodium and potassium cations than to chloride or phosphate anions. A bacterial cross-feeding assay was used to demonstrate that Vpu can also form sodium-permeable channels in vivo in the E. coli plasma membrane.
Induction of apoptosis by baculovirus transactivator IE1
Authors: Prikhod'ko E A , Miller L K
Published: January 2020

Apoptosis is induced upon infection of SF-21 cells by mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) lacking a functional p35 gene which encodes a stoichiometric inhibitor of members of the interleukin-1beta converting enzyme family of cysteine proteases (N.J. Bump et al, Science 269:1885-1888, 1995; R.J. Clem, M. Fechheimer, and L.K. Miller, Science 254:1388-1390, 1991). We found that transfection of SF-21 cells with the AcMNPV ie-1 gene was sufficient to induce apoptosis, which was characterized by fragmentation of cellular DNA into oligonucleosomal fragments and apoptotic body formation. No signs of apoptosis were observed in Trichoplusia ni TN-368 cells transfected with ie-1, a result which is consistent with the observation that p35 mutants of AcMNPV do not induce apoptosis in this cell line. Cotransfection of SF-21 cells with p35 blocked ie-1-induced apoptosis, indicating that expression of ie-1 activates apoptosis through a p35-inhibitable cysteine protease pathway. Cotransfection with Cp-iap, an active member of another family of antiapoptotic inhibitors of apoptosis (iaps), also inhibited IE1-induced apoptosis. Thus, ie-1 may participate in inducing apoptosis in AcMNPV-infected cells, although the dependence of induction on DNA replication suggests that ie-1 is not the direct apoptotic signal during infection. The ie-1 gene product, IE1, is known to be a potent transactivator of baculovirus gene expression that interacts with specific palindromic sequences which can act as transcriptional enhancers and as origins of DNA replication in transient assays.
Characterization of hepatitis C virus replication in cloned cells obtained from a human T-cell leukemia virus type 1-infected cell line, MT-2
Authors: Mizutani T , Kato N , Saito S , Ikeda M , Sugiyama K , Shimotohno K
Published: January 2020

We recently found that a human T-cell leukemia virus type 1-infected cell line, MT-2, could support the replication of hepatitis C virus (HCV) (N. Kato, T. Nakazawa, T. Mizutani, and K. Shimotohno, Biochem. Biophys. Res. Commun. 206:863-869, 1995). In order to develop a culture system in which HCV replicates more efficiently, we examined the efficiency of HCV replication in cloned MT-2 cell lines by the limiting dilution method. Consequently, we obtained five clones in which intracellular positive-stranded HCV RNA could be detected until at least 21 days postinoculation (p.i.), as opposed to 15 days p.i. in uncloned MT-2 cells. MT-2C, one of the five clones which supported HCV replication up to 30 days p.i., was used for further characterization of HCV replication. Semiquantitative analysis of HCV by PCR revealed that RNA synthesis in infected cells increased after inoculation, reached a maximum level at 4 days p.i., and maintained this level until at least 11 days p.i. The 5' untranslated region of negative-stranded HCV RNA was also detected in the infected cells by two different methods with strand specificity. These results suggest that HCV replicated and multiplied in the MT-2C cells. HCV-infected MT-2C cells that were treated with antibiotics, such as G418 and hygromycin B, sustained HCV RNA for a longer period than did untreated cells. We demonstrated inhibitory effects on HCV replication by an antisense oligonucleotide complementary to the HCV core encoding region and by interferon-alpha. Furthermore, cell-free viral transmission was demonstrated by this culture system. These results suggest that our cell culture system will be useful for studying the mechanism of HCV replication, for screening antiviral agents, and for developing HCV vaccines.
Clonal propagation of Epstein-Barr virus (EBV) recombinants in EBV-negative Akata cells
Authors: Shimizu N , Yoshiyama H , Takada K
Published: January 2020

We lack a host cell supporting an efficient lytic replication of Epstein-Barr virus (EBV). Recently, we isolated EBV-negative cell clones from the Akata cell line (referred as Akata- [N. Shimizu, A. Tanabe-Tochikura, Y. Kuroiwa, and K. Takada, J. Virol. 68:6069-6073, 1994). Since the parental Akata line is one of the highest EBV producers, we examined whether Akata- cells had become a good host for EBV propagation. The parental Akata cells have about 20 copies of EBV plasmid per cell. A drug resistance gene was inserted into one of them by homologous recombination. The resultant virus preparation, a mixture of wild-type and recombinant EBV, was used to infect Akata- cells. After incubation in the selective medium, drug-resistant Akata- cell clones were isolated and proved to be infected with recombinant EBV only. By treatment of the cells with antiimmunoglobulin antibodies, a large amount of recombinant EBV (i.e., more than 10 microg/1-liter culture) was produced. In contrast, three other B-lymphoma lines, BJAB, Ramos, and Louckes, were nonpermissive for virus replication. These results indicate that Akata- cells are suitable for propagation of recombinant EBV clonally, which becomes a powerful tool for determining EBV genetics and which makes it possible to use EBV as a vector for gene therapy.
The varicella-zoster virus ORF66 protein induces kinase activity and is dispensable for viral replication
Authors: Heineman T C , Seidel K , Cohen J I
Published: January 2020

Varicella-zoster virus (VZV) open reading frames (ORFs) 47 and 66 encode proteins that are homologous to a family of eukaryotic serine-threonine kinases. Prior studies showed that the VZV ORF47 protein has kinase activity in vitro and is dispensable for replication in cultured cells. To examine the role of the ORF66 protein during infection, we constructed VZV recombinants that are unable to express either the ORF66 protein (ROka 66S) or both the ORF47 and ORF66 proteins (ROka 47S/66S). VZV unable to express ORF66 grew to titers similar to those of the parental VZV (ROka) in vitro; however, VZV lacking both ORF66 and ORF47 grew to titers lower than those of ROka. Nuclear extracts from ROka 66S- or ROka 47S-infected cells showed a 48-kDa phosphoprotein(s); a phosphoprotein with a similar size was not present in nuclear extracts from ROka 47S/66S-infected cells. To determine the role of the ORF66 protein in the phosphorylation of specific VZV-encoded proteins, we immunoprecipitated known VZV phosphoproteins (ORF4, ORF62, ORF63, and ORF68 proteins) from nuclear extracts of phosphate-labeled cells infected with ROka, ROka 66S, or ROka 47S/66S. Each of the VZV phosphoproteins was phosphorylated to a similar extent in the presence or absence of either the ORF66 protein or both the ORF66 and ORF47 proteins. From these studies we conclude (i) neither ORF66 alone nor ORF66 and ORF47 in combination are essential for VZV growth in cultured cells, (ii) ORF66 either is a protein kinase or induces protein kinase activity during infection, and (iii) the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 do not require either ORF66 alone or ORF66 and ORF47 for phosphorylation in vitro.
Infection of primary CD4+ and CD8+ T lymphocytes by Epstein-Barr virus enhances human immunodeficiency virus expression
Authors: Guan M , Zhang R D , Wu B , Henderson E E
Published: January 2020

CD4+ and CD8+ T lymphocytes purified from normal adult donors by flow cytometry could be infected with Epstein-Barr virus (EBV) as measured by the accumulation of components of the EBV replicative cycle, viral DNA and viral transcripts encoding EBER1 and BRLF1. EBV infection resulted in enhanced replication of human immunodeficiency virus type 1 (HIV-1) IIIB in CD4+ lymphocytes as measured by accumulation of reverse transcriptase and formation of syncytia. Furthermore, a small percentage of CD8+ T cells became permissive after infection with EBV. Inactivation of transforming functions by irradiation with UV light greatly reduced the ability of EBV to enhance HIV-1 replication in T4+ T cell, suggesting that live virus is needed for enhancement. These results demonstrate a direct synergy between EBV and HIV-1 during coinfection of T cells in vitro and may explain the beneficial effect of acyclovir in combination with antiretroviral chemotherapy as well as the increased incidence of T-cell lymphomas associated with EBV in patients with AIDS.
Identification of the major membrane and core proteins of vaccinia virus by two-dimensional electrophoresis
Authors: Jensen O N , Houthaeve T , Shevchenko A , Cudmore S , Ashford T , Mann M , Griffiths G , Krijnse Locker J
Published: January 2020

Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known about the molecular events that occur during that process. Towards this goal, we have identified the major membrane and core proteins of the intracellular mature virus (IMV). Pure IMV preparations were subjected to Nonidet P-40 (NP-40) and dithiothreitol (DTT) treatment to separate the core proteins from the membrane proteins. These proteins were subsequently separated by two-dimensional (2D) gel electrophoresis, and the major polypeptide spots, as detected by silver staining and 35S labeling, were identified by either matrix-assisted laser desorption/ionization mass spectrometry, N-terminal amino acid sequencing, or immunoprecipitation with defined antibodies. Sixteen major spots that partitioned into the NP-40-DTT-soluble fraction were identified; 11 of these were previously described virally encoded proteins and 5 were cellular proteins, mostly of mitochondrial origin. The core fraction revealed four major spots of previously described core proteins, two of which were also detected in the membrane fraction. Subsequently, the NP-40-DTT-soluble and -insoluble fractions from purified virus preparations, separated by 2D gels, were compared with postnuclear supernatants of infected cells that had been metabolically labeled at late times (6 to 8 h) postinfection. This relatively short labeling period as well as the apparent shutoff of host protein synthesis allowed the selective detection in such postnuclear supernatants of virus-encoded proteins. These postnuclear supernatants were subsequently treated with Triton X-114 or with sodium carbonate to distinguish the membrane proteins from the soluble proteins. We have identified the major late membrane and nonmembrane proteins of the IMV as they occur in the virus as well as in infected cells. This 2D gel map should provide an important reference for future molecular studies of vaccinia virus morphogenesis.
Residue Trp-48 of Tva is critical for viral entry but not for high-affinity binding to the SU glycoprotein of subgroup A avian leukosis and sarcoma viruses
Authors: Zingler K , Young J A
Published: January 2020

Previously, mutant Tva receptors were classified as either partially or completely defective in mediating subgroup A avian leukosis and sarcoma virus (ALSV-A) entry (C. Bélanger, K. Zingler, and J. A. T. Young, J. Virol. 69:1019-1024, 1995; K. Zingler, C. Bélanger, R. Peters, D. Agard, and J. A. T. Young, J. Virol. 69:4261-4266, 1995). To specifically test the abilities of these mutant Tva proteins to bind ALSV-A surface (SU) protein, binding studies were performed with a subgroup A SU-immunoadhesin. This fusion protein is composed of the subgroup A Schmidt-Ruppin SU protein fused in frame to a rabbit immunoglobulin constant region. This reagent was conjugated to fluorescein isothiocyanate and used for flow cytometric analysis with transfected human 293 cells expressing different forms of Tva. The SU-immunoadhesin bound the wild-type Tva protein with a KD of approximately 1.5 nM. Amino acid substitutions that reduced viral entry at Asp-46 and at Cys-35 and Cys-50, which are predicted to form an intrachain disulfide bond in Tva, drastically reduced the binding affinity for the SU-immunoadhesin. Thus, the effects on viral entry of some mutations could be explained solely by changes in the binding affinity for ALSV-A SU. However, this was not true for other mutations tested, especially those with amino acid substitutions that replaced Trp-48. Compared with the wild-type receptor, these latter mutations led to approximately 43- to 200-fold reductions in viral infectivity but only to approximately 2.5- to 3.4-fold reductions in the binding affinity for the SU-immunoadhesin. These results support a role for Trp-48 of Tva in mediating steps of viral entry subsequent to binding ALSV-A SU.
Anergic TH1 clones specific for hepatitis B virus (HBV) core peptides are inhibitory to other HBV core-specific CD4+ T cells in vitro
Authors: Diepolder H M , Jung M C , Wierenga E , Hoffmann R M , Zachoval R , Gerlach T J , Scholz S , Heavner G , Riethmüller G , Pape G R
Published: January 2020

A strong and transient hepatitis B virus core (HBc)-specific CD4+ T-cell response has been shown to be associated with viral elimination in acute self-limited hepatitis B but to be absent in chronic hepatitis B. So far, little is known about immunological mechanisms involved in the regulation of the HBc-specific CD4+ T-cell response. We studied 28 patients with acute hepatitis B, and frequently a sudden decrease in the HBc-specific CD4+ T-cell response was found between 4 and 8 weeks after disease onset. Thirty-two CD4+ T-cell clones specific for amino acids 50 to 69, 81 to 105, 117 to 131, or 141 to 165 of HBc were isolated from a patient shortly before the peripheral blood mononuclear cell response to most HBc-derived peptides abruptly disappeared. TH1 clones, but not TH0 clones, could be anergized in vitro by stimulation with specific peptides even in the presence of costimulatory cells. Moreover, when anergic cells were mixed with responsive cells, the proliferation of HBc-specific TH1 or TH0 clones was inhibited antigen specifically by anergic cells. The unusual susceptibility of HBc-specific TH1 clones to anergy induction in vitro as well as their potential to inhibit other HBc-specific TH1 and TH0 clones suggests that anergy induction may be involved in the downregulation of the virus-specific immune response during acute hepatitis B in vivo.
Tracking hepatitis C virus quasispecies major and minor variants in symptomatic and asymptomatic liver transplant recipients
Authors: Gretch D R , Polyak S J , Wilson J J , Carithers R L , Perkins J D , Corey L
Published: January 2020

To evaluate the possibility that distinct viral quasispecies play a role in the pathogenesis of progressive hepatitis C virus (HCV) infection, we performed a detailed evaluation of HCV quasispecies before and after liver transplantation in five patients infected with HCV genotype 1, three of whom developed severe recurrent hepatitis C and two of whom developed asymptomatic posttransplant infections with high-titered viremia. HCV quasispecies were characterized by using a combination of nucleotide sequencing plus heteroduplex tracking assay of the second envelope gene hypervariable region (HVR). An average of 30 HVR clones were analyzed per specimen; an average of five specimens were analyzed per patient over a 6- to 24-month study period. The complexity of HCV quasispecies in pretransplant serum varied, ranging from one to nine genetically distinct variants for the five patients. However, in all five cases, relatively homogenous quasispecies variants emerged after liver transplantation. In the three patients who developed recurrent hepatitis, quasispecies major variants present in pretransplant serum were efficiently propagated immediately after liver transplantation and were propagated throughout the course of acute and chronic hepatitis. In contrast, in the two asymptomatic cases, we observed rapid depletion of pretransplant quasispecies major variants from posttransplant serum, followed by emergence of new quasispecies variants by posttransplant day 30. Genetic analysis suggested that in these cases, the new quasispecies variants were derived from minor variants present at relatively low clonal frequency (less than 5% of HVR clones) within the pretransplant quasispecies populations. These data demonstrate that quasispecies tracking patterns are associated with the rapidity and severity of HCV-associated liver disease after liver transplantation. Further characterization of HCV quasispecies in animal model systems is warranted.
Cytotoxic T-lymphocyte responses to cytomegalovirus in normal and simian immunodeficiency virus-infected rhesus macaques
Authors: Kaur A , Daniel M D , Hempel D , Lee-Parritz D , Hirsch M S , Johnson R P
Published: January 2020

Disseminated cytomegalovirus (CMV) infection is a frequent occurrence in human immunodeficiency virus-infected humans and in simian immunodeficiency virus (SIV)-infected rhesus macaques. Rhesus macaques are a suitable animal model with which to study in vivo interactions between CMV and AIDS-associated retroviruses. Since cytotoxic T lymphocytes (CTL) play a major role in control of viral infections, we have characterized CMV-specific CTL responses in SIV-infected and uninfected rhesus macaques. Autologous fibroblasts infected with rhesus CMV were used to stimulate freshly isolated peripheral blood mononuclear cells from CMV-seropositive animals. Following in vitro stimulation, specific CTL activity against CMV-infected autologous fibroblasts was detected in CMV-seropositive but not in CMV-seronegative normal macaques. CMV-specific CTL activity comparable to that in normal animals was also detected in two CMV-seropositive macaques infected with a live attenuated SIV strain (SIVdelta3) and in two of three macaques infected with pathogenic SIV strains. The CMV-specific CTL response was class I major histocompatibility complex restricted and mediated by CD8+ cells. An early CMV protein(s) was the dominant target recognized by bulk CTL, although the pattern of CTL recognition of CMV proteins varied among animals. Analysis of CMV-specific CTL responses in macaques should serve as a valuable model for CMV immunopathogenesis and will facilitate prospective in vivo studies of immune interactions between CMV and SIV in AIDS.