bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational analysis of the hepatitis C virus RNA helicase
Authors: Kim D W , Kim J , Gwack Y , Han J H , Choe J
Published: January 2020

The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. A serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed.
Direct interaction of hepatitis C virus core protein with the cellular lymphotoxin-beta receptor modulates the signal pathway of the lymphotoxin-beta receptor
Authors: Chen C M , You L R , Hwang L H , Lee Y H
Published: January 2020

Previous studies suggest that the core protein of hepatitis C virus (HCV) has a pleiotropic function in the replication cycle of the virus. To understand the role of this protein in HCV pathogenesis, we used a yeast two-hybrid protein interaction cloning system to search for cellular proteins physically interacting with the HCV core protein. One such cellular gene was isolated and characterized as the gene encoding the lymphotoxin-beta receptor (LT-betaR). In vitro binding analysis demonstrated that the HCV core protein binds to the C-terminal 98 amino acids within the intracellular domain of the LT-betaR that is involved in signal transduction, although the binding affinity of the full-length HCV core protein was weaker than that of its C-terminally truncated form. Our results also indicated that the N-terminal 40-amino-acid segment of the HCV core protein was sufficient for interaction with LT-betaR and that the core protein could form complexes with the oligomeric form of the intracellular domain of LT-betaR, which is a prerequisite for downstream signaling of this receptor. Similar to other members of the tumor necrosis factor (TNF) receptor superfamily, LT-betaR is involved in the cytotoxic effect of the signaling pathway, and thus we have elucidated the biological consequence of interaction between the HCV core protein and LT-betaR. Our results indicated that in the presence of the synergizing agent gamma interferon, the HCV core protein enhances the cytotoxic effects of recombinant forms of LT-betaR ligand in HeLa cells but not in hepatoma cells. Furthermore, this enhancement of the cytolytic activity was cytokine specific, since in the presence of cycloheximide, the expression of the HCV core protein did not elicit an increase in the cytolytic activity of TNF in both HeLa and hepatoma cells. In summary, the HCV core protein can associate with LT-betaR, and this protein-protein interaction has a modulatory effect on the signaling pathway of LT-betaR in certain cell types. Given the known roles of LT-betaR/LT-alpha1,beta2 receptor-ligand interactions in the normal development of peripheral lymphoid organs and in triggering cytolytic activity and NF-kappaB activation in certain cell types, our finding implies that the HCV core protein may aggravate these biological functions of LT-betaR, resulting in pathogenesis in HCV-infected cells.
Alveolar macrophages regulate the induction of primary cytotoxic T-lymphocyte responses during influenza virus infection
Authors: Wijburg O L , DiNatale S , Vadolas J , van Rooijen N , Strugnell R A
Published: January 2020

Virus-specific cytotoxic T lymphocytes (CTL) are thought to be responsible for the eradication of respiratory influenza virus infections by direct cytolysis of virus-infected epithelial cells. In this study, we provide evidence for a role for alveolar macrophages (AM) in the regulation of pulmonary virus-specific CTL responses. Prior to infection with influenza virus, AM were selectively eliminated in vivo with a liposome-mediated depletion technique, and virus-specific CTL activities of lung and mediastinal lymph node (MLN) cells were assayed ex vivo and compared with those for normal mice. AM depletion resulted in increased primary CTL responses and changed the kinetics of the CTL response. Flow cytometric analysis of lung and MLN cells showed that the percentage of CD8+ cells was not altered after AM depletion and that lung cells from AM-depleted mice had an increased capacity to lyse virus-infected cells. Upon restimulation in vitro, virus-specific CTL activity in lung cells of normal mice was similar to that in lung cells of AM-depleted mice. Furthermore, elimination of AM resulted in increased virus titers in the lung, but virus clearance as a function of time was not affected. Our results show that AM regulate virus-specific CTL responses during respiratory influenza virus infection by removing viral particles, by downregulating the priming and activity of CTL in MLN cells, and by inhibiting the expansion of virus-specific CTL in the lung.
Activation of the adenovirus major late promoter by transcription factors MAZ and Sp1
Authors: Parks C L , Shenk T
Published: January 2020

Multiple binding sites for the transcription factors MAZ and Sp1 within the adenovirus type 5 major late promoter have been identified by DNase I protection studies. In the proximal region of the promoter, both MAZ and Sp1 interact with GC-rich sequences flanking the TATA box. Two MAZ binding sites are centered at -18 and -36 relative to the transcriptional initiation site. Sp1 bound only to the -18 GC-rich sequence. Several sites of interaction were also evident in the distal region of the promoter. Both MAZ and Sp1 interacted with a sequence centered at -166, and MAZ bound weakly to an additional site centered at -130. Overexpression of MAZ or Sp1 activated expression from the major late promoter in transient expression assays. Mutational analysis of the GC-rich sequences in the major late promoter suggested that a primary target of MAZ activation is the GC-rich sequences flanking the TATA sequence, whereas Sp1 requires the distal GC-rich sequence elements to stimulate gene expression. This activation is enhanced by the adenovirus E1A protein, and evidence for interaction between E1A and both transcription factors was obtained by using an immunoprecipitation assay. Activation by MAZ and Sp1 also was observed in transfection studies using the complete adenovirus type 5 genome as the target. Increased levels of late mRNA from both the L1 and L5 regions were observed when MAZ or Sp1 expression plasmids were transfected with viral DNA. Unexpectedly, activation of the major late promoter by MAZ and Sp1 was detected irrespective of whether the viral DNA could replicate.
Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome
Authors: Patton J T , Jones M T , Kalbach A N , He Y W , Xiaobo J
Published: January 2020

Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis.
Dissecting the roles of VP0 cleavage and RNA packaging in picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus
Authors: Curry S , Fry E , Blakemore W , Abu-Ghazaleh R , Jackson T , King A , Lea S , Newman J , Stuart D
Published: January 2020

Empty capsids of foot-and-mouth disease virus (FMDV) type A22 Iraq 24/64, whose structure has been solved by X-ray crystallography, are unusual for picornaviruses since they contain VP2 and VP4, the cleavage products of the protein precursor VP0. Both the N terminus of VP1 and the C terminus of VP4, which pack together close to the icosahedral threefold symmetry axis where three pentamers associate, are more disordered in the empty capsid than they are in the RNA-containing virus. The ordering of these termini in the presence of RNA strengthens interactions within a single protomer and between protomers belonging to different pentamers. The disorder in the FMDV empty capsid forms a subset of that seen in the poliovirus empty capsid, which has VP0 intact. Thus, VP0 cleavage confers stability on the picornavirus capsid over and above that attributable to RNA encapsidation. In both FMDV and poliovirus empty capsids, the internal disordering uncovers a conserved histidine which has been proposed to be involved in the cleavage of VP0. A comparison of the putative active sites in FMDV and poliovirus suggests a structural explanation for the sequence specificity of the cleavage reaction.
Allele-specific adaptation of poliovirus VP1 B-C loop variants to mutant cell receptors
Authors: Liao S , Racaniello V
Published: January 2020

Previous work has shown that three different mutations in domain 1 of the poliovirus receptor (Pvr), two in the predicted C'-C" ridge and one in the D-E loop, abolish binding of the P1/Mahoney strain. All three receptor defects could be suppressed by a mutation in the VP1 B-C loop of the viral capsid that was present in all 16 P1/Mahoney isolates adapted to the mutant receptors. To identify allele-specific mutations that enable poliovirus to utilize mutant receptors, and to understand the role of the VP1 B-C loop in adaptation, we selected mutant receptor-adapted viruses derived from two P1/Mahoney variants, one which lacks the VP1 B-C loop and one in which the VP1 B-C loop is replaced with the corresponding sequence from the P2/Lansing strain. Six adapted viral isolates were obtained after passage on mutant receptor-expressing cell lines. Sequence analysis revealed that each virus contained three to five mutations, and a total of 18 amino acid changes at 17 capsid residues were identified. Site-directed mutagenesis was used to evaluate the role of these mutations in adaptation to mutant Pvr. The results demonstrate that mutations in the viral canyon floor and rim are allele specific and compensate only for receptor defects in the C'-C" ridge of Pvr, suggesting that these sites interact in the virus-receptor complex. Furthermore, mutations in the VP1 E-F loop suppressed Pvr D-E loop defects, implying that the Pvr D-E loop contacts the VP1 E-F loop. Most of the other mutations mapped to interior capsid residues, some interacting with the fivefold- or threefold-related protomers. These mutations may regulate receptor interaction by controlling the structural flexibility of the viral capsid. In viruses lacking the VP1 B-C loop, single mutations were not sufficient to confer the adapted phenotype, in contrast to the 414 virus, which contains the B-C loop. Although the VP1 B-C loop appeared to be dispensable for adaptation, it may have provided a selective advantage in adaptation of P1/Mahoney to mutant Pvr.
A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells
Authors: Shafren D R , Williams D T , Barry R D
Published: January 2020

The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.
Role of a single amino acid at the amino terminus of the simian virus 5 F2 subunit in syncytium formation
Authors: Ito M , Nishio M , Kawano M , Kusagawa S , Komada H , Ito Y , Tsurudome M
Published: January 2020

The fusion (F) protein of simian virus 5 (strain W3A) induced extensive cell fusion in BHK cells when expressed alone, while that of strain WR did not. Mutational analysis demonstrated that the fusing activity can be transferred to the WR F protein by a proline residue at position 22 of subunit W3A F2.
Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex
Authors: Rubenwolf S , Schütt H , Nevels M , Wolf H , Dobner T
Published: January 2020

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa EIB protein can exert its multiple activities in lytically infected cells and in adenovirus transformation.
Nuclear sites of herpes simplex virus type 1 DNA replication and transcription colocalize at early times postinfection and are largely distinct from RNA processing factors
Authors: Phelan A , Dunlop J , Patel A H , Stow N D , Clements J B
Published: January 2020

We have visualized the intracellular localization of herpes simplex virus (HSV) type 1 replication and transcription sites in infected HeLa cells by using direct labelling methods. The number of viral transcription foci increases in a limited way; however, the number of replication sites increases in a near-exponential manner throughout infection, and both replication and transcription sites are found buried throughout the nuclear interior. Simultaneous visualization of viral transcription and replication foci shows that the two processes colocalize at early times, but at later times postinfection, there are additional sites committed solely to replication. This contrasts with the situation in adenovirus-infected cells in which, throughout replication, sites of transcription are adjacent to but do not colocalize with sites of viral DNA replication. The data for an increase in HSV transcription sites suggest an initial phase of replication of input genomes which are then transcribed. Sites of HSV replication colocalize with viral DNA replication and packaging proteins but are largely distinct from the punctate distribution of small nuclear ribonucleoprotein particles. Very high multiplicities of infection have shown an upper limit of some 18 viral transcription foci per nucleus, suggesting cellular constraints on transcription site formation. Use of virus replication mutants confirms that the labelled foci are sites of viral RNA and DNA synthesis; in the absence of viral DNA replication functions, no replication foci and only a limited number of transcription foci were present. Absence of a packaging function had no apparent effect on transcription or replication site formation, illustrating that DNA packaging is not a prerequisite for ongoing DNA synthesis. Further, the essential HSV protein IE63 is required for efficient replication site formation at later times postinfection but is not required for transcription foci formation.
Early cytokine and chemokine gene expression in lymph nodes of macaques infected with simian immunodeficiency virus is predictive of disease outcome and vaccine efficacy
Authors: Zou W , Lackner A A , Simon M , Durand-Gasselin I , Galanaud P , Desrosiers R C , Emilie D
Published: January 2020

Competitive PCR was used to evaluate the expression of cytokine, granzyme B, and chemokine genes in lymph nodes of macaques recently infected with the simian immunodeficiency virus (SIV) pathogenic molecular clone SIVmac239 (n = 16), the nonpathogenic vaccine strain SIVmac239 delta nef (n = 8), and the nonpathogenic molecular clone SIVmac1A11 (n = 8). For both SIVmac239 and its nef-deleted derivative, strong expression was observed as early as 7 days postinfection for interleukin 1beta (IL-1beta), IL-6, tumor necrosis factor alpha, gamma interferon, and IL-13. The levels of gene induction were equally intense for both viruses despite a lower viral load for SIVmac239 deltanef compared with that for SIVmac239. However, the nature of the cytokine network activation varied with the viral inocula. Primary infection with SIVmac239 was characterized by a higher level of IL-4, IL-10, MIP-1alpha, MIP-1beta, MCP-1, and RANTES gene expression and a lower level of IL-12 and granzyme B gene expression compared with infection with SIVmac239 delta nef. Thus, infection with nef-deleted SIV was associated with a preferential Th1 versus Th2 pattern of cytokine production. Infection with SIVmac1A11 was characterized by a delayed immune response for all markers tested. The unique patterns of cytokine and chemokine gene expression in lymph nodes correlated nicely with the pathogenic potential of the SIV strains used as well as with differences in their ability to serve as protective vaccines.
S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus
Authors: Oleksiewicz M B , Alexandersen S
Published: January 2020

We examined replication of the autonomous parvovirus Aleutian mink disease parvovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle arrest occurred exclusively in cells containing de novo-synthesized viral nonstructural (NS) proteins. Production of ADV NS proteins, indicative of ADV replication, was triggered during S-phase traverse. The NS+ cells that were generated during later parts of S phase did not undergo cytokinesis and formed a distinct population, termed population A. Formation of population A was not prevented by VM-26, indicating that these cells were arrested in late S or G2 phase. Cells in population A continued to support high-level ADV DNA replication and production of infectious virus after the normal S phase had ceased. A second, postmitotic, NS+ population (termed population B) arose in G0/G1, downstream of population A. Population B cells were unable to traverse S phase but did exhibit low-level DNA synthesis. Since the nature of this DNA synthesis was not examined, we cannot at present differentiate between G1 and early S arrest in population B. Cells that became NS+ during S phase entered population A, whereas population B cells apparently remained NS- during S phase and expressed high NS levels postmitosis in G0/G1. This suggested that population B resulted from leakage of cells with subthreshold levels of ADV products through the late S/G2 block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell.
In vivo study of genetically simplified bovine leukemia virus derivatives that lack tax and rex
Authors: Boris-Lawrie K , Altanerova V , Altaner C , Kucerova L , Temin H M
Published: January 2020

Genetically simplified derivatives of complex retroviruses that replicate in animal models are useful tools to study the role of the complex regulatory genes in virus infection and pathogenesis and were proposed as a novel approach toward the development of vaccines against complex retroviruses. Previously we developed genetically simple derivatives of bovine leukemia virus (BLV) that can replicate in tissue culture independently of the BLV regulatory proteins, Tax and Rex, and the RIII and GIV open reading frames (K. Boris-Lawrie and H. M. Temin, J. Virol. 69:1920-1924, 1995). These derivatives are encoded on novel, hybrid retrovirus genomes that contain transcriptional control sequences of a simple retrovirus and gag-pol or env genes of the complex BLV. The first-generation simple BLV derivatives replicate as complementary viruses (coviruses) by using separate gag-pol or env genomes, and therefore virus spread is limited to cells that are infected with both covirus genomes. Here we describe a second-generation simple BLV derivative that is encoded on a single hybrid genome. We show the virus to be replication competent by successive passage on D17 target cells and by analysis of viral RNA and proteins in the infected cells. Furthermore, we evaluate the immunogenicity and infectivity of the simple BLV derivatives in a BLV animal model. Small groups of rats were injected either with virus-producing cells or with proviral DNA. Western immunoblot analysis revealed that antibodies against the major viral antigenic determinants are induced in response to either method of introduction and that seroconversion is sustained in most of the rats for at least 6 months (the duration of the study). The magnitudes of the antiviral responses were similar in rats infected with the first-generation simple BLV coviruses, the second-generation replication-competent derivative, or wild-type BLV. Wild-type BLV typically infects peripheral blood mononuclear cells (PBMC), and the simple BLV derivatives were also found to infect PBMC as demonstrated by PCR amplification of proviral sequences and reverse transcriptase PCR amplification of viral RNA in treated rats. These results establish that simple BLV derivatives lacking tax and rex are infectious and immunogenic in rats. These viruses will be useful tools in comparative studies with BLV to evaluate the role of tax and rex in maintenance of virus load and in disease outcome.
Human immunodeficiency virus type 1 gp120 stimulates cytomegalovirus replication in monocytes: possible role of endogenous interleukin-8
Authors: Capobianchi M R , Barresi C , Borghi P , Gessani S , Fantuzzi L , Ameglio F , Belardelli F , Papadia S , Dianzani F
Published: January 2020

Recombinant gp120, but not other human immunodeficiency type 1 (HIV-1) structural proteins, dose-dependently stimulates human cytomegalovirus (HCMV) immediate-early antigen (IEA) expression and infectious virus yield in freshly isolated normal monocytes infected with HCMV. Monoclonal antibodies (MAbs) recognizing the gp120 V3 loop, as well as V3 loop octameric multibranched peptides and antibody to galactocerebroside, but not sCD4, abrogate the gp120 stimulation of IEA expression, suggesting that the effect involves V3 loop-galactocerebroside interaction and is not mediated by CD4. Interleukin 8 (IL-8) gene expression is enhanced in monocytes treated with gp120 at the level of both mRNA and released protein. Exogenous IL-8 could replace gp120 in the stimulation of HCMV infection, while a MAb capable of neutralizing IL-8 activity abrogates the gp120-induced HCMV stimulation. These data indicate that HIV-1 glycoprotein induces stimulation of productive infection of monocytes with HCMV and that such stimulation may be mediated by the upregulation of IL-8 gene expression. This is the first evidence that HIV-1 may affect HCMV replication indirectly, via the interaction of gp120 with the monocyte membrane, in the complete absence of retroviral replication, through the stimulation of IL-8 release. Because in HIV-1-infected individuals, HCMV infection is frequently activated and the levels of circulating IL-8 are enhanced, these findings may be pathogenetically relevant.
Persistent poliovirus infection in mouse motoneurons
Authors: Destombes J , Couderc T , Thiesson D , Girard S , Wilt S G , Blondel B
Published: January 2020

Poliovirus (PV) is the causal agent of paralytic poliomyelitis. Many survivors of the acute disease, after decades of clinical stability, develop new muscular symptoms called postpolio syndrome. It has been hypothesized that the persistence of PV in the spinal cord is involved in the etiology of this syndrome. To investigate the ability of PV to persist in the spinal cord after the onset of paralysis, we exploited a mouse model in which most animals inoculated with a mouse-adapted mutant survived after the onset of paralysis. Light microscopy and ultrastructural immunohistochemical studies and reverse transcription followed by nested PCR performed on spinal cord from paralyzed mice demonstrated that PV persisted in the mouse spinal cord for at least 12 months after the onset of paralysis. This mouse model provides a new tool for studying poliomyelitis evolution after the onset of paralysis.
Human cytomegalovirus infection inhibits G1/S transition
Authors: Dittmer D , Mocarski E S
Published: January 2020

Cell cycle progression during cytomegalovirus infection was investigated by fluorescence-activated cell sorter (FACS) analysis of the DNA content in growth-arrested as well as serum-stimulated human fibroblasts. Virus-infected cells maintained in either low (0.2%) or high (10%) serum failed to progress into S phase and failed to divide. DNA content analysis in the presence of G1/S (hydroxyurea and mimosine) and G2/M (nocodazole and colcemid) inhibitors demonstrated that upon virus infection of quiescent (G0) cells, the cell cycle did not progress beyond the G1/S border even after serum stimulation. Proteins which normally indicate G1/S transition (proliferating cell nuclear antigen [PCNA]) or G2/M transition (cyclin B1) were elevated by virus infection. PCNA levels were induced in infected cells and exhibited a punctate pattern of nuclear staining instead of the diffuse pattern observed in mock-infected cells. Cyclin B1 was induced in infected cells which exhibited a G1/S DNA content by FACS analysis, suggesting that expression of this key cell cycle function was dramatically altered by viral functions. These data demonstrate that contrary to expectations, cytomegalovirus inhibits normal cell cycle progression. The host cell is blocked prior to S phase to provide a favorable environment for viral replication.
Expression and maturation of human foamy virus Gag precursor polypeptides
Authors: Giron M L , Colas S , Wybier J , Rozain F , Emanoil-Ravier R
Published: January 2020

In this report, we address the processing of the Gag polypeptides of human foamy virus previously reported to be atypical. In the cytoplasm or the nucleus of infected cells as well as in free virus particles, two Gag precursor polypeptides were identified at approximately 72 and 68 kDa, p72 giving rise to p68 by a maturation process. Efficient maturation of Gag precursors was observed only in two situations: (i) during the early steps of virus adsorption and (ii) under experimental conditions, including treatment with DNase I, known to dissociate actin polymers associated with high ionic strength and ionic detergents. Rather than being a defective viral protease function, an association of Gag precursors with a cytoskeleton network might be responsible for the low rate of Gag protein maturation through inhibition of their cleavage by the protease.
Antibody cross-reactivity with CD46 and lack of cell surface expression suggest that moesin might not mediate measles virus binding
Authors: Devaux P , Gerlier D
Published: January 2020

The binding of antimoesin antibodies from ascites fluids to the surfaces of human and rodent cells was found to parallel the level of CD46 expression. No such reactivity was detected with a purified antimoesin antibody which recognized intracellular moesin. In Western blots, antimoesin antibodies were found to react with solubilized CD46 and a recombinant soluble form of CD46. Antimoesin antibodies also reacted with CD46/CD4 molecules containing only the SCR I and II domains required for measles virus (MV) hemagglutinin binding onto CD46. We suggest that the weak cross-reactivity of antimoesin antibodies with CD46 explains the inhibitory effect of these antibodies on MV entry and that moesin is not directly involved in MV binding.
Inhibition of human immunodeficiency virus fusion by a monoclonal antibody to a coreceptor (CXCR4) is both cell type and virus strain dependent
Authors: McKnight A , Wilkinson D , Simmons G , Talbot S , Picard L , Ahuja M , Marsh M , Hoxie J A , Clapham P R
Published: January 2020

CXCR4 (also termed fusin, LESTR, or HUMSTR) is a member of the G-protein-coupled chemokine receptor family with seven membrane-spanning domains. CXCR4 acts as a coreceptor for syncytium-inducing human immunodeficiency virus type 1 (HIV-1) strains, conferring entry into CD4+ cells. We show here that a novel mouse monoclonal antibody (12G5) that recognizes CXCR4 blocked cell-to-cell fusion and cell free-virus infection of CXCR4+ CD4+ RD rhabdomyosarcoma cells by seven HIV-1 and HIV-2 strains that had various cell tropisms for different CD4+ human cell types. Yet the majority of the members of the same virus panel resisted 12G5 inhibition on T-cell lines. When inhibition was observed on these cell types, it was both cell type and virus strain dependent. In at least one situation, 12G5 failed to block LAI infection of cells expressing CXCR4 as the only available coreceptor. Our observations suggest that CXCR4 could be processed or presented differently depending on the cell type, allowing some strains to evade 12G5 inhibition. Alternatively, since several of the viruses could infect certain CXCR4- CD4+ cell lines, it is conceivable that alternative coreceptors are active, enabling individual HIV strains to choose between compatible coreceptors during entry into cells. Moreover, the strain dependency of 12G5 inhibition implies that the interaction of different HIVs with CXCR4 varies.
Induction of CD4 expression and human immunodeficiency virus type 1 replication by mutants of the interferon-inducible protein kinase PKR
Authors: Nagai K , Wong A H , Li S , Tam W N , Cuddihy A R , Sonenberg N , Mathews M B , Hiscott J , Wainberg M A , Koromilas A E
Published: January 2020

Replication of the human immunodeficiency virus type 1 (HIV-1) is inhibited by interferons (IFNs), and the IFN-inducible protein kinase PKR is thought to mediate this effect by regulating protein synthesis. Here we report that ectopic expression of dominant negative PKR mutants in Jurkat cells induces HIV-1 replication. Specifically, expression of CD4 is upregulated by the PKR mutants, and this correlates with an induction of HIV-1 binding and proviral DNA synthesis upon HIV-1 infection. Moreover, activation of NF-kappaB was induced by an RNA binding-defective mutant of PKR. Thus, it appears that PKR, in addition to translational control, is involved in HIV-1 replication by modulating virus binding through the regulation of CD4 expression and virus gene expression through the activation of NF-kappaB.
Infectious properties of human immunodeficiency virus type 1 mutants with distinct affinities for the CD4 receptor
Authors: Platt E J , Madani N , Kozak S L , Kabat D
Published: January 2020

Recent evidence suggests that primary patient isolates of T-cell-tropic human immunodeficiency virus type 1 (HIV-1 ) have lower affinities for CD4 than their laboratory-adapted derivatives, that this may partly result from tighter gp120-gp41 bonds that constrain the CD4 binding sites of the primary viruses, and that selection for increased CD4 affinity may be the principal factor in laboratory adaptation of HIV-1 (S. L. Kozak, E. J. Platt, N. Madani, F. E. Ferro, Jr., K. Peden, and D. Kabat, J. Virol. 71:873-882, 1997). These conclusions were based on studies with a panel of HeLa-CD4 cell clones that differ in CD4 levels over a broad range, with laboratory-adapted viruses infecting all clones with equal efficiencies and primary T-cell-tropic viruses infecting the clones in proportion to cellular CD4 levels. Additionally, all of the primary and laboratory-adapted T-cell-tropic viruses efficiently used CXCR-4 (fusin) as a coreceptor. To test these conclusions by an independent approach, we studied mutations in the laboratory-adapted virus LAV/IIIB that alter the CD)4 binding region of gp120 and specifically reduce CD4 affinities of free gp 120 by 85 to 98% (U. Olshevsky et al., J. Virol. 64:5701-5707, 1990). These mutations reduced virus titers to widely varying extents that ranged from severalfold to several orders of magnitude and converted infectivities on the HeLa-CD4 panel from CD4 independency to a high degree of CD4 dependency that resembled the behavior of primary patient viruses. The relative infectivities of the mutants correlated closely with their sensitivities to inactivation by soluble CD4 but did not correlate with the relative CD4 affinities of their free gp120s. Most of the mutations did not substantially alter envelope glycoprotein synthesis, processing, expression on cell surfaces, incorporation into virions, or rates of gp120 shedding from virions. However, one mutation (D457R) caused a decrease in gp160 processing by approximately 80%. The fact that several mutations increased rates of spontaneous viral inactivation (especially D368P) suggests that HIV-1 life spans may be determined by structural stabilities of viral envelope glycoproteins. All of the wild-type and mutant viruses were only slowly and inefficiently adsorbed onto cultured CD4-positive cells at 37 degrees C, and the gradual declines in viral titers in the media were caused almost exclusively by spontaneous inactivation rather than by adsorption. The extreme inefficiency with which infectious HIV-1 is able to infect cultured susceptible CD4-positive cells in standard assay conditions casts doubt on previous inferences that the vast majority of retrovirions produced in cultures are noninfectious. Apparent infectivity of T-cell-tropic HIV-1 in culture is limited by productive associations with CD4 and is influenced in an interdependent manner by CD4 affinities of viral gp120-gp41 complexes and quantities of cell surface CD4.
Genetic exchange by recombination or reassortment is infrequent in natural populations of a tripartite RNA plant virus
Authors: Fraile A , Alonso-Prados J L , Aranda M A , Bernal J J , Malpica J M , García-Arenal F
Published: January 2020

Two hundred seventeen field isolates of cucumber mosaic cucumovirus (CMV), sampled from 11 natural populations, were typed by RNase protection assay (RPA) using probes from the genomic RNAs of strains in subgroup I and in subgroup II of CMV strains. Most (85%) of the analyzed isolates belonged to subgroup I. For these subgroup I isolates, only two clearly different RPA patterns, A and B, were found for each of four probes representing RNA1, RNA2, and each of the two open reading frames in RNA3. On the basis of these RPA patterns for each probe, different haplotypes were defined. The frequency composition for these haplotypes differed for the various analyzed populations, with no correlation with place or year of sampling. This genetic structure corresponds to a metapopulation with local extinctions and recolonizations. Most subgroup I isolates (73%) belonged to haplotypes with RPA pattern A (type 1) or B (type 2) for all four probes. A significant fraction of subgroup I isolates (16%) gave evidence of mixed infections with these two main types, from which genetic exchange could occur. Genetic exchange by segment reassortment was seen to occur: the fraction of reassortant isolates was 4%, reassortment did not occur at random, and reassortants did not become established in the population. Thus, there is evidence of selection against reassortment between types 1 and 2 of subgroup I isolates. Aphid transmission experiments with plants doubly infected with type 1 and type 2 isolates gave further evidence that reassortment is selected against in CMV. Genetic exchange by recombination was detected for RNA3, for which two RPA probes were used. Recombinant isolates amounted to 7% and also did not become established in CMV populations. Sequence analyses of regions of RNA1, RNA2, and RNA3 showed that there are strong constraints to maintain the encoded sequence and also gave evidence that these constraints may have been different during divergence of types 1 and 2 and, later on, during diversification of these two types. Constraints to the evolution of encoded proteins may be related to selection against genetic exchange. Our data, thus, do not favor current hypotheses that explain the evolution of multipartite viral genomes to promote genetic exchange.
Protease-deficient herpes simplex virus protects mice from lethal herpesvirus infection
Authors: Hippenmeyer P J , Rankin A M , Luckow V A , Neises G R
Published: January 2020

Null mutants and attenuated mutants of herpes simplex virus (HSV) have been shown to induce immunity against challenge from wild-type virus. Null viruses with a defect in late gene products would be expected to express more viral genes than viruses with defects in essential early gene products and thus induce a better immune response. Herpesviruses encode a late gene product (serine protease) that is autocatalytic and cleaves the capsid assembly protein during viral replication. To determine whether a virus with a mutation in this gene could induce immunity, we constructed a recombinant virus containing the gusA reporter gene in the protease domain of the HSV type 1 UL26 open reading frame (ORF). Consistent with previous results (M. Gao, L. Matusick-Kumar, W. Hurlburt, S. F. DiTusa, W. W. Newcomb, J. C. Brown, P. J. McCann, I. Deckman, and R. J. Colonno, J. Virol. 68:3702-3712, 1994), recombinant virus could be isolated only from helper cell lines expressing the product of the UL26 ORF. Mice inoculated with the recombinant virus were unaffected by doses of virus that were lethal to mice infected with wild-type virus. Mice which were previously inoculated with the recombinant virus were also protected by a subsequent challenge with wild-type virus in a dose-dependent manner. These results indicate that recombinant viruses lacking the protease gene are avirulent but render protection from subsequent challenge.
The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication
Authors: Fischer F , Peng D , Hingley S T , Weiss S R , Masters P S
Published: January 2020

The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. Both alterations created silent changes in the N reading frame. In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque size in comparison with its isogenic wild-type counterpart, suggesting that expression of I confers some minor growth advantage to the virus. The engineered mutations were stable during the course of experimental infection in mice, and the I mutant showed no significant differences from wild type in its ability to replicate in the brains or livers of infected animals. These results demonstrate that I protein is not essential for the replication of MHV either in tissue culture or in its natural host.
The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter
Authors: Pereira D J , Muzyczka N
Published: January 2020

Control of adeno-associated virus (AAV) transcription from the three AAV promoters (p5, p19, and p40) requires the adenovirus E1a protein and the AAV nonstructural (Rep) proteins. The Rep proteins have been shown to repress the AAV p5 promoter yet facilitate activation of the p19 and p40 promoters during a productive infection. To elucidate the mechanism of promoter regulation by the AAV Rep proteins, the cellular factors involved in mediating Rep activation of the p19 promoter were characterized. A series of protein-DNA binding experiments using extracts derived from uninfected HeLa cells was performed to identify cellular factors that bind to the p19 promoter. Electrophoretic mobility shift assays, DNase I protection analyses, and UV cross-linking experiments demonstrated specific interactions with the cellular factor SP1 (or an SP1-like protein) at positions -50 and -130 relative to the start of p19 transcription. Additionally, an unknown cellular protein (cellular AAV activating protein [cAAP]) with an approximate molecular mass of 34 kDa was found to interact with a CArG-like element at position -140. Mutational analysis of the p19 promoter suggested that the SP1 site at -50 and the cAAP site at -140 were necessary to mediate Rep activation of p19. Antibody precipitation experiments demonstrated that Rep-SP1 protein complexes can exist in vivo. Although Rep was demonstrated to interact with p19 DNA directly, the affinity of Rep binding was much lower than that seen for the Rep binding elements within the terminal repeat and the p5 promoter. Furthermore, the interaction of purified Rep68 with the p19 promoter in vitro was negligible unless purified SP1 was also added to the reaction. Thus, the ability of Rep to transactivate the p19 promoter is likely to involve SP1-Rep protein contacts that facilitate Rep interaction with p19 DNA.
Role of the intergenic dinucleotide in vesicular stomatitis virus RNA transcription
Authors: Barr J N , Whelan S P , Wertz G W
Published: January 2020

To investigate the role played by the intergenic dinucleotide sequence of the conserved vesicular stomatitis virus (VSV) gene junction in modulation of polymerase activity, we analyzed the RNA synthesis activities of bicistrionic genomic analogs that contained either the authentic N/P gene junction or gene junctions that had been altered to contain either the 16 possible dinucleotide combinations, single nucleotide intergenic sequences, or no intergenic sequence at all. Quantitative measurements of the amounts of upstream, downstream, and readthrough mRNAs that were transcribed by these mutant templates showed that the behavior of the viral polymerase was profoundly affected by the nucleotide sequence that it encountered as it traversed the gene junction, although the polymerase was able to accommodate a remarkable degree of sequence variation without altogether losing the ability to terminate and reinitiate transcription. Alteration or removal of the intergenic sequence such that the U tract responsible for synthesis of the upstream mRNA poly(A) tail was effectively positioned adjacent to the consensus downstream gene start signal resulted in almost complete abrogation of downstream mRNA synthesis, thus defining the intergenic sequence as an essential sequence element of the gene junction. Many genome analogs with altered intergenic sequences directed abundant synthesis of a readthrough transcript without correspondingly high levels of downstream mRNA, an observation inconsistent with the shunting model of VSV transcription, which suggests that polymerase molecules are prepositioned at gene junctions, awaiting a push from upstream. Instead, the findings of this study support a model of sequential transcription in which initiation of downstream mRNA can occur only following termination of the preceding transcript.