bluuebunny/crossref_metadata_2025 huggingface dataset:
Approaches to determine stoichiometry of viral assembly components
Authors: Trottier M , Guo P
Published: January 2020

Due to the rapidity of biological reactions, it is difficult to isolate intermediates or to determine the stoichiometry of participants in intermediate reactions. Instead of determining the absolute amount of each component, this study involved the use of relative parameters, such as dilution factors, percentages probabilities, and slopes of titration curves, that can be more accurately quantified to determine the stoichiometry of components involved in bacteriophage phi29 assembly. This work takes advantage of the sensitive in vitro phage phi29 assembly system, in which 10(8) infectious virions per ml without background can be assembled from eight purified components. It provides a convenient assay for quantification of the stoichiometry of packaging components, including the viral procapsid, genomic DNA, DNA-packaging pRNA, and other structural proteins and enzymes. The presence of a procapsid binding domain and another essential functional domain within the pRNA makes it an ideal component for constructing lethal mutants for competitive procapsid binding. Two methods were used for stoichiometry determination. Method 1 was to determine the combination probability of mutant and wild-type pRNAs bound to procapsids. The probability of procapsids that possess a certain amount of mutant and a certain amount of wild-type pRNA, both with an equal binding affinity, was predicted with the binomial equation [EQUATION IN TEXT] where Z is the total number of pRNAs per procapsid, M is the number of mutant pRNAs bound to one procapsid, and (ZM) is equal to [FORMULA IN TEXT]. With various ratios of mutant to wild-type pRNA in in vitro viral assembly, the percent mutant pRNA versus the yield of virions was plotted and compared to a series of predicted curves to find a best fit. It was determined that five or six copies of pRNA were required for one DNA-packaging event, while only one mutant pRNA per procapsid was sufficient to block packaging. Method 2 involved the comparison of slopes of curves of dilution factors versus the yield of virions. Components with known stoichiometries served as standard controls. The larger the stoichiometry of the component, the more dramatic the influence of the dilution factor on the reaction. A slope of 1 indicates that one copy of the component is involved in the assembly of one virion. A slope larger than 1 would indicate multiple-copy involvement. By this method, the stoichiometry of gp11 in phi29 particles was determined to be approximately 12. These approaches are useful for the determination of the stoichiometry of functional units involved in viral assembly, be they single molecules or oligomers. However, these approaches are not suitable for the determination of exact copy numbers of individual molecules involved if the functional unit is composed of multiple subunits prior to assembly.
Tyrosine phosphorylation events during coxsackievirus B3 replication
Authors: Huber M , Selinka H C , Kandolf R
Published: January 2020

In order to study cellular and viral determinants of pathogenicity, interactions between coxsackievirus B3 (CVB3) replication and cellular protein tyrosine phosphorylation were investigated. During CVB3 infection of HeLa cells, distinct proteins become phosphorylated on tyrosine residues, as detected by the use of antiphosphotyrosine Western blotting. Two proteins of 48 and 200 kDa showed enhanced tyrosine phosphorylation 4 to 5 h postinfection (p.i.), although virus-induced inhibition of cellular protein synthesis had already occurred 3 to 4 h p.i. Subcellular fractionation experiments revealed distinct localization of tyrosine-phosphorylated proteins of 48 and 200 kDa in the cytosol and membrane fractions of infected cells, respectively. In addition, in Vero cells infected with CVB3, echovirus (EV)11, or EV12, increased tyrosine phosphorylation of a 200-kDa protein was detected 6 h p.i. Herbimycin A, a specific inhibitor of Src-like protein tyrosine kinases, was shown to inhibit virus-induced tyrosine phosphorylations and to reduce the production of progeny virions. In contrast, in cells treated with the inhibitors staurosporine and calphostin C, the synthesis of progeny virions was not affected. Immunoprecipitation experiments suggested that the tyrosine-phosphorylated 200-kDa protein in CVB3-infected cells is of cellular origin. In summary, these investigations have begun to unravel the effect of CVB3 as well as EV11 and EV12 replication on cellular tyrosine phosphorylation and support the importance of tyrosine phosphorylation events for effective virus replication. Such cellular phosphorylation events triggered in the course of enterovirus infection may enhance virus replication.
Virus-specific memory and effector T lymphocytes exhibit different cytokine responses to antigens during experimental murine respiratory syncytial virus infection
Authors: Srikiatkhachorn A , Braciale T J
Published: January 2020

Mice sensitized to the G (attachment) or F (fusion) glycoproteins of respiratory syncytial virus (RSV) expressed different patterns of cytokine production and lung pathology when challenged by intranasal infection with RSV. Five days after challenge, mice sensitized to G glycoprotein produced high levels of interleukin-4 (IL-4) and IL-5 in the lungs and spleens and developed extensive pulmonary eosinophilia, while mice sensitized to F glycoprotein produced IL-2 and developed a mononuclear cell infiltration. Memory lymphocytes isolated 2 weeks after intranasal challenge of mice primed to the G or F glycoprotein secreted only IL-2 and gamma interferon (IFN-gamma) when stimulated with RSV. IL-4 and IL-5 production characteristic of Th2-type effectors in the lung was observed only after multiple rounds of in vitro stimulation of RSV G-specific memory T lymphocytes with antigen. Also IFN-gamma production appeared to play only a minor role in the expression of pulmonary pathology characteristic of Th1 or Th2 T-lymphocyte responses, because mice genetically deficient in IFN-gamma production by gene disruption displayed the same pattern of pulmonary inflammation to RSV infection after priming to RSV F or G as conventional mice. These results suggest that effector T lymphocytes exhibit a different pattern of cytokine production than memory T-lymphocyte precursors precommitted to a Th1 or Th2 pattern of differentiation. Furthermore, these observations raise the possibility that the cytokine response of human memory T lymphocytes after a single exposure to antigen in vitro may not accurately reflect the cytokine response of differentiated effector T lymphocytes at the site of infection in vivo.
Formation of native hepatitis C virus glycoprotein complexes
Authors: Deleersnyder V , Pillez A , Wychowski C , Blight K , Xu J , Hahn Y S , Rice C M , Dubuisson J
Published: January 2020

The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.
Susceptibility of nude mice carrying the Fv-4 gene to Friend murine leukemia virus infection
Authors: Higo K , Kubo Y , Iwatani Y , Ono T , Maeda M , Hiai H , Masuda T , Kuribayashi K , Zhang F , Lamin T Y , Adachi A , Ishimoto A
Published: January 2020

Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). Despite complete resistance to ecotropic MuLV infection in mice carrying the Fv-4 gene, it is known that cells carrying the resistance gene in tissue culture do not always show resistance as extensive as that in vivo (H. Yoshikura and T. Odaka, JNCI 61:461-463, 1978). To investigate the immunological effect on resistance in vivo, we introduced the Fv-4 gene into BALB/c nude mice (Fv-4-/- nude[nu/nu]) by mating them with Fv-4 congenic BALB/c mice (Fv-4r/r nude+/+) and examined the susceptibility of the F2 progeny to F-MuLV. All BALB/c nude mice without the Fv-4 gene (Fv-4-/- nude[nu/nu]) were permissive to F-MuLV and developed erythroleukemia within 2 weeks after virus inoculation. The BALB/c nude mice with the Fv-4 gene (Fv-4r/r nude[nu/nu]) did not develop leukemia, and no or little virus was detected in the spleen 7 weeks after virus inoculation. The resistance to F-MuLV was dominant in (Fv-4 congenic BALB/c x BALB/c nude) F1 mice with the Fv-4r/- nude(nu/+) genotype as strictly as in (Fv-4 congenic BALB/c x BALB/c) F1 mice with the Fv-4r/- nude+/+ genotype. However, almost all BALB/c nude mice with the Fv-4r/- nude(nu/nu) genotype developed the disease within 7 weeks, and the virus was detected in all of their spleens even in the mice without leukemia. These results show that the resistance caused by the Fv-4 gene is recessive in nude mice and dominant in BALB/c mice. Some immunological effects, perhaps cell-mediated immunity, may play important roles in the resistance to F-MuLV infection in vivo in addition to the dosage effect of the Fv-4 product.
Sequences flanking the core DNA-binding domain of bovine papillomavirus type 1 E2 contribute to DNA-binding function
Authors: Pepinsky R B , Prakash S S , Corina K , Grossel M J , Barsoum J , Androphy E J
Published: January 2020

We have compared a series of molecular constructs that contain the minimal DNA-binding and dimerization domain of bovine papillomavirus type 1 (BPV-1) E2 alone or this binding domain plus the adjacent 16 or 40 amino acids to test the role of the flanking sequences in E2 function. The presence of these sequences resulted in an up to eightfold increase in the affinity of E2 for its target DNA and stabilized the protein against denaturation both in the absence of DNA and in the form of DNA-protein complexes. In addition, an aspartic acid-to-tyrosine mutation within the flanking region blocked DNA binding and function. These data demonstrate that sequences flanking the core domain contribute to E2 function and are, in fact, an integral part of the DNA-binding domain of BPV-1 E2.
Viral determinants of rotavirus pathogenicity in pigs: production of reassortants by asynchronous coinfection
Authors: Tauscher G I , Desselberger U
Published: January 2020

A porcine rotavirus (prv), variant 4F, isolated in tissue culture from the feces of a Chinese pig with diarrhea, was found to have become highly pathogenic when passaged in gnotobiotic piglets (J. C. Bridger, B. Burke, G. M. Beards, and U. Desselberger, J. Gen. Virol. 73:3011-3015, 1992). Comparison with the closely related pig-apathogenic variant prv 4S suggested the outer capsid protein VP4 (encoded by RNA 4) of prv 4F as a determinant for pathogenicity (B. Burke, J. C. Bridger, and U. Desselberger, J. Gen. Virol. 75:2205-2212, 1994; B. Burke, J. C. Bridger, and U. Desselberger, Virology 202:754-759, 1994). In order to provide more direct evidence, the pathogenic prv 4F variant which grows and forms plaques poorly in tissue culture was reassorted with the well-tissue-culture-adapted, pig-apathogenic bovine rotavirus (brv; UK Compton strain). After asynchronous coinfection of cell cultures (first prv 4F, followed by brv 6 to 12 h later), several reassortants were isolated containing RNA 4 of prv 4F either alone (isolate B-F4) or together with one or two other genes of 4F in the genetic background of brv. Backcrossing of the monoreassortant B-F4 with prv 4S yielded a monoreassortant, S-F4, which carries RNA 4 of the 4F variant in the genetic background of prv 4S. The in vitro growth characteristics of these reassortants were analyzed, and the roles of VP4 in plaque formation and growth kinetics in cell culture were confirmed. The monoreassortant S-F4 and the parental viruses prv 4F and prv 4S are currently being tested for pathogenicity in gnotobiotic piglets (J. C. Bridger, G. Tauscher, and U. Desselberger, unpublished data).
Mutational analysis of the virion host shutoff gene (UL41) of herpes simplex virus (HSV): characterization of HSV type 1 (HSV-1)/HSV-2 chimeras
Authors: Everly D N , Read G S
Published: January 2020

During lytic herpes simplex virus (HSV) infections, the half-lives of host and viral mRNAs are regulated by the HSV virion host shutoff (Vhs) protein (UL41). The sequences of the UL41 polypeptides of HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical. In spite of this similarity, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. To examine type-specific differences in Vhs function, we compared the Vhs activities of UL41 alleles from HSV-1(KOS) and HSV-2(333) by assaying the ability of a transfected UL41 allele to inhibit expression of a cotransfected reporter gene. Both HSV-1 and HSV-2 alleles inhibited reporter gene expression over a range of vhs DNA concentrations. However, 40-fold less of the HSV-2 allele was required to yield the same level of inhibition as HSV-1, indicating that it is significantly more potent. Examination of chimeric UL41 alleles containing various combinations of HSV-1 and HSV-2 sequences identified three regions of the 333 polypeptide which increase the activity of KOS when substituted for the corresponding amino acids of the KOS protein. These are separated by two regions which have no effect on KOS activity, even though they contain 43 of the 74 amino acid differences between the parental alleles. In addition, alleles encoding a full-length KOS polypeptide with a 32-amino-acid N-terminal extension retain considerable activity. The results begin to identify which amino acid differences are responsible for type-specific differences in Vhs activity.
Increased lymphomagenicity and restored disease specificity of AML1 site (core) mutant SL3-3 murine leukemia virus by a second-site enhancer variant evolved in vivo
Authors: Ethelberg S , Lovmand J , Schmidt J , Luz A , Pedersen F S
Published: January 2020

SL3-3 is a highly T-lymphomagenic murine retrovirus. The major genetic determinant of disease is the transcriptional enhancer, which consists of a repeated region with densely packed binding sites for several transcription factors, including AML1 (also known as core binding factor and polyoma enhancer-binding protein 2) and nuclear factor 1 (NF1). Previously, we examined the enhancer structure of proviruses from murine tumors induced by SL3-3 with mutated AML1 (core) sites and found a few cases of second-site alterations. These consisted of deletions involving the NF1 sites and alterations in overall number of repeat elements, and they conferred increased enhancer strength in transient transcription assays. We have now tested the pathogenicity of a virus harboring one such second-site variant enhancer in inbred NMRI mice. It induced lymphomas with a 100% incidence and a significantly shorter latency than the AML1 mutant it evolved from. The enhancer structure thus represents the selection for a more tumorigenic virus variant during the pathogenic process. Sequencing of provirus from the induced tumors showed the new enhancer variant to be genetically stable. Also, Southern blotting showed that the tumors induced by the variant were T-cell lymphomas, as were the wild-type-induced lymphomas. In contrast, tumors induced by the original core/AML1 site I-II mutant appeared to be of non-T-cell origin and several proviral genomes with altered enhancer regions could be found in the tumors. Moreover, reporter constructs with the new tumor-derived variant could not be transactivated by AML1 in cotransfection experiments as could the wild type. These results emphasize the importance of both core/AML1 site I and site II for the pathogenic potential of SL3-3 and at the same time show that second-site alterations can form a viral variant with a substantial pathogenic potential although both AML1 sites I and II are nonfunctional.
A highly sensitive in vitro infection assay to explore early stages of mouse mammary tumor virus infection
Authors: Vacheron S , Renno T , Acha-Orbea H
Published: January 2020

Mouse mammary tumor virus (MMTV) infection of adult mice induces a strong response to superantigen (Sag) in their draining lymph nodes, which results from the presentation of Sag by MMTV-infected B cells to Sag-reactive T cells. To date, infection with physiologically relevant doses of MMTV can be detected in vivo only after several days of Sag-mediated T-cell-dependent amplification of infected B cells. Furthermore, no efficient in vitro system of detecting MMTV infection is available. Such a model would allow the dissection of the early phase of infection, the assessment of the contributions of different cell types, and the screening of large panels of molecules for their potential roles in infection and Sag response. For these reasons, we have established an in vitro model for detecting infection which is as sensitive and reproducible as the in vivo model. We found that the viral envelope (Env) protein is crucial for target cell infection but not for presentation of Sag. Furthermore, we show that infection of purified B cells with MMTV induces entry of Sag-responsive T cells into the cell cycle, while other professional antigen-presenting cells, such as dendritic cells, are much less efficient in inducing a response.
Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal-deleted VP2: implications for the architecture of the VP2 capsid layer
Authors: Lawton J A , Zeng C Q , Mukherjee S K , Cohen J , Estes M K , Prasad B V
Published: January 2020

Rotaviruses are the leading cause of severe infantile gastroenteritis worldwide. These viruses are large, complex icosahedral particles consisting of three concentric capsid layers enclosing a genome of eleven segments of double-stranded RNA (dsRNA). The amino terminus of the innermost capsid protein VP2 possesses a nonspecific single-stranded RNA and dsRNA binding activity, and the amino terminus is also essential for the incorporation of the polymerase enzyme VP1 and guanylyltransferase VP3 into the core of the virion. Biochemical and structural studies have suggested that VP2, and especially the amino terminus, appears to act as a scaffold for proper assembly of the components of the viral core. To locate the amino terminus of VP2 within the core, we have used electron cryomicroscopy and image reconstruction to determine the three-dimensional structures of recombinant virus-like particles that contain either full-length or amino-terminal-deleted forms of VP2 coexpressed with the intermediate capsid protein VP6. A comparison of these structures indicates two significant changes along the inner surface of VP2 in the structure lacking the amino terminus: a loss of mass adjacent to the fivefold axes and a redistribution of mass along the fivefold axes. Examination of the VP2 layer suggests that the proteins are arranged as dimers of 120 quasi-equivalent molecules, with each dimer extending between neighboring fivefold axes. Our results indicate that the amino termini of both quasi-equivalent VP2 molecules are located near the icosahedral vertices.
Inhibition of parvovirus minute virus of mice replication by a peptide involved in the oligomerization of nonstructural protein NS1
Authors: Pujol A , Deleu L , Nüesch J P , Cziepluch C , Jauniaux J C , Rommelaere J
Published: January 2020

The large nonstructural protein NS1 of the minute virus of mice and other parvoviruses is involved in essential steps of the viral life cycle, such as DNA replication and transcriptional regulation, and is a major contributor to the toxic effect on host cells. Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to NS1. Homo-oligomerization is a prerequisite for a number of proteins to be fully functional. In particular, helicases generally act as homo-oligomers. Indirect evidence of NS1 self-association has been recently obtained by a nuclear cotransport assay (J. P. Nüesch and P. Tattersall, Virology 196:637-651, 1993). In order to demonstrate the oligomerizing property of NS1 in a direct way and localize the protein region(s) involved, the yeast two-hybrid system was used in combination with deletion mutagenesis across the whole NS1 molecule, followed by high-resolution mapping of the homo-oligomerization domain by a peptide enzyme-linked immunosorbent assay method. This study led to the identification of a distinct NS1 peptide that contains a bipartite domain involved in NS1 oligomerization. Furthermore, this isolated peptide was found to act as a specific competitive inhibitor and suppress NS1 helicase activity in vitro and parvovirus DNA replication in vivo, arguing for the involvement of NS1 oligomerization in these processes. Our results point to drug targeting of oligomerization motifs of viral regulatory proteins as a potentially useful antiviral strategy.
Viral dynamics of primary viremia and antiretroviral therapy in simian immunodeficiency virus infection
Authors: Nowak M A , Lloyd A L , Vasquez G M , Wiltrout T A , Wahl L M , Bischofberger N , Williams J , Kinter A , Fauci A S , Hirsch V M , Lifson J D
Published: January 2020

Mathematical modeling of viral replication dynamics, based on sequential measurements of levels of virion-associated RNA in plasma during antiretroviral treatment, has led to fundamental new insights into human immunodeficiency virus type 1 pathogenesis. We took advantage of the simian immunodeficiency virus (SIV)-infected macaque model to perform detailed measurements and mathematical modeling during primary infection and during treatment of established infection with the antiretroviral drug (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). The calculated clearance half-life for productively infected cells during resolution of the peak viremia of primary infection was on the order of 1 day, with slightly shorter clearance half-lives calculated during PMPA treatment. Viral reproduction rates upon discontinuation of PMPA treatment after 2 weeks were approximately twofold greater than those obtained just prior to initiation of treatment in the same animals, likely reflecting accumulation of susceptible target cells during treatment. The basic reproductive ratio (R0) for the spread of SIV infection in vivo, which represents the number of productively infected cells derived from each productively infected cell at the beginning of infection, was also estimated. This parameter quantifies the extent to which antiviral therapy or vaccination must limit the initial spread of virus to prevent establishment of chronic disseminated infection. The results thus provide an important guide for efforts to develop vaccines against SIV and, by extension, human immunodeficiency virus.
Cre/loxP-mediated excision of a neomycin resistance expression unit from an integrated retroviral vector increases long terminal repeat-driven transcription in human hematopoietic cells
Authors: Fernex C , Dubreuil P , Mannoni P , Bagnis C
Published: January 2020

Recombinant retroviruses are currently the most attractive vehicles for gene transfer into hematopoietic cells. Retroviral vectors often contain an easily selectable marker gene in addition to the gene of interest. However, the presence and selection for expression of the selectable gene often result in a significant reduction of the expression of the gene of interest in the transduced cells. In order to circumvent this problem, we have developed a Cre/loxP recombination system for specific excision of the selectable expression unit from integrated retroviruses. A retroviral vector, containing both a neomycin resistance expression unit flanked by loxP sites and granulocyte-macrophage colony-stimulating factor cDNA, was used to transduce the human hematopoietic K-562 cell line. Four transduced cell clones were then superinfected with a retrovirus containing a Cre recombinase expression unit. Molecular analyses of 30 doubly transduced subclones showed a strict correlation between cre expression and loxP-flanked selectable cassette excision, thus implying that Cre recombinase activity is very efficient in a retroviral context. Moreover, the excision of the selectable cassette results in a significant increase of granulocyte-macrophage colony-stimulating factor transcription driven by the retroviral promoter.
A bulged stem-loop structure in the 3' untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication
Authors: Hsue B , Masters P S
Published: January 2020

The 3' untranslated region (UTR) of the positive-sense RNA genome of the coronavirus mouse hepatitis virus (MHV) contains sequences that are necessary for the synthesis of negative-strand viral RNA as well as sequences that may be crucial for both genomic and subgenomic positive-strand RNA synthesis. We have found that the entire 3' UTR of MHV could be replaced by the 3' UTR of bovine coronavirus (BCV), which diverges overall by 31% in nucleotide sequence. This exchange between two viruses that are separated by a species barrier was carried out by targeted RNA recombination. Our results define regions of the two 3' UTRs that are functionally equivalent despite having substantial sequence substitutions, deletions, or insertions with respect to each other. More significantly, our attempts to generate an unallowed substitution of a particular portion of the BCV 3' UTR for the corresponding region of the MHV 3' UTR led to the discovery of a bulged stem-loop RNA secondary structure, adjacent to the stop codon of the nucleocapsid gene, that is essential for MHV viral RNA replication.
Expression of the Autographa californica nuclear polyhedrosis virus apoptotic suppressor gene p35 in nonpermissive Spodoptera littoralis cells
Authors: Gershburg E , Rivkin H , Chejanovsky N
Published: January 2020

Apoptosis was postulated as the main barrier to replication of the Autographa californica nuclear polyhedrosis virus (AcMNPV) in a Spodoptera littoralis SL2 cell line (N. Chejanovsky and E. Gershburg, Virology 209:519-525, 1995). Thus, we hypothesized that the viral apoptotic suppressor gene p35 is either poorly expressed or nonfunctional in AcMNPV-infected SL2 cells. These questions were addressed by first determining the steady-state levels of the p35 product, P35, in AcMNPV-infected SL2 cells. Indeed, very low levels of P35 were found in infected SL2 cells in comparison with those in SF9 cells. Overexpression of p35, in transient-transfection and recombinant-virus infection experiments, inhibited actinomycin D- and AcMNPV-induced apoptosis, as determined by reduced cell blebbing and release of oligonucleosomes and increased cell viability of SL2. However, SL2 budded-virus (BV) titers of a recombinant AcMNPV which highly expressed p35 did not improve significantly. Also, injection of S. littoralis larvae with recombinant and wild-type AcMNPV BVs showed similar 50% lethal doses. These data suggest that apoptosis is not the only impediment to AcMNPV replication in these nonpermissive S. littoralis cells, and probably in S. littoralis larvae, so p35 may not be the only host range determinant in this system.
Fungal phosphate transporter serves as a receptor backbone for gibbon ape leukemia virus
Authors: Pedersen L , van Zeijl M , Johann S V , O'Hara B
Published: January 2020

Pit1, the receptor for gibbon ape leukemia virus (GALV), is proposed to be an integral membrane protein with five extracellular loops. Chimeras made between Pit1 homologs differing in permissivity for infection and between Pit1 and the related protein Pit2 have shown that the fourth extracellular loop plays a critical role in infection. However, further elucidation of the roles of the extracellular loops in infection is hampered by the high level of sequence similarity among these proteins. The sodium-dependent phosphate transporter, Pho-4, from the filamentous fungus Neurospora crassa is distantly related to Pit1 and -2, showing an amino acid identity of only 35% to Pit1 in the putative extracellular loops. We show here that Pho-4 itself does not function as a receptor for GALV. Introduction of 12 Pit1-specific amino acid residues in the putative fourth extracellular loop of Pho-4 resulted in a functional GALV receptor. Therefore, the presence of a Pit1 loop 4-specific sequence is sufficient to confer receptor function for the mammalian retrovirus GALV on the fungal phosphate transporter Pho-4.
Foot-and-mouth disease virus and poliovirus particles contain proteins of the replication complex
Authors: Newman J F , Brown F
Published: January 2020

Nonstructural proteins 2C, 3CD, 3C, and 3D, and the cellular protein actin, are present in highly purified preparations of foot-and-mouth disease virus (FMDV) and poliovirus. They remain bound in variable amounts to the RNAs when the RNAs are extracted from the viruses with phenol or phenol-sodium dodecyl sulfate (SDS) and, for FMDV, when the RNA is released from the particles by a lowering of the pH below 7. RNA prepared by these methods is rapidly degraded at 37 degrees C, particularly in the presence of NH4+ ions, but hydrolysis can be prevented by antibody against Escherichia coli-expressed 3D, indicating that it is the RNA polymerase that has nuclease activity. In contrast, virion RNA from which the nonstructural proteins and actin have been removed by extraction with guanidine thiocyanate-phenol-chloroform or proteinase K-phenol is stable at 37 degrees C, although its specific infectivity is lower than that of the RNA extracted with phenol or phenol-SDS. The possible implications of the close association of replication complex proteins with the RNA in virus particles are discussed.
Incorporation of functional human immunodeficiency virus type 1 integrase into virions independent of the Gag-Pol precursor protein
Authors: Liu H , Wu X , Xiao H , Conway J A , Kappes J C
Published: January 2020

Retroviral integrase (IN) is expressed and incorporated into virions as part of the Gag-Pol polyprotein precursor. IN catalyzes integration of the proviral DNA into host cell chromosomes during the early stages of the virus life cycle, and as a component of Gag-Pol, it is involved in virion morphogenesis during late stages. It is unknown whether the scheme, conserved among retroviruses, for expressing and incorporating IN as a component of the Gag-Pol precursor protein is necessary for its function in the infected cell after viral entry. We have developed human immunodeficiency virus (HIV) virion-associated accessory proteins (Vpr and Vpx) as vehicles to deliver both foreign and viral proteins into the virus particle by their expression in trans as heterologous fusion proteins (X. Wu, et al., J. Virol. 69:3389-3398, 1995; X. Wu, et al., J. Virol. 70:3378-3384, 1996; X. Wu, et al., EMBO J. 16:5113-5122, 1977). To analyze IN function independent of its expression as a part of Gag-Pol, we expressed and incorporated IN into HIV type 1 (HIV-1) virions in trans as a fusion partner of Vpr (Vpr-IN). Our results demonstrate that the Vpr-IN fusion protein is efficiently incorporated into virions and then processed by the viral protease to liberate the IN protein. Virus derived from IN-minus provirus is noninfectious. However, this defect is overcome by trans complementation with the Vpr-IN fusion protein. Moreover, complemented virions are able to replicate through a complete cycle of infection, including formation of the provirus (integration). These results show, for the first time, that full IN function can be provided in trans, independent of its expression and incorporation into virions as a component of Gag-Pol. This finding also indicates that the IN domain of Gag-Pol is not required for the formation of infectious virions when IN is provided in trans. The ability to incorporate functional IN into retroviral particles in trans will provide unique opportunities to explore the function of this critical enzyme in a biologically relevant context, i.e., in infected cells as part of the nucleoprotein/preintegration complex.
Coronavirus genomic and subgenomic minus-strand RNAs copartition in membrane-protected replication complexes
Authors: Sethna P B , Brian D A
Published: January 2020

The majority of porcine transmissible gastroenteritis coronavirus plus-strand RNAs (genome and subgenomic mRNAs), at the time of peak RNA synthesis (5 h postinfection), were not found in membrane-protected complexes in lysates of cells prepared by Dounce homogenization but were found to be susceptible to micrococcal nuclease (85%) or to sediment to a pellet in a cesium chloride gradient (61%). They therefore are probably free molecules in solution or components of easily dissociable complexes. By contrast, the majority of minus-strand RNAs (genome length and subgenomic mRNA length) were found to be resistant to micrococcal nuclease (69%) or to remain suspended in association with membrane-protected complexes following isopycnic sedimentation in a cesium chloride gradient (85%). Furthermore, 35% of the suspended minus strands were in a dense complex (1.20 to 1.24 g/ml) that contained an RNA plus-to-minus-strand molar ratio of approximately 8:1 and viral structural proteins S, M, and N, and 65% were in a light complex (1.15 to 1.17 g/ml) that contained nearly equimolar amounts of plus- and minus-strand RNAs and only trace amounts of proteins M and N. In no instance during fractionation were genome-length minus strands found segregated from sub-genome-length minus strands. These results indicate that all minus-strand species are components of similarly structured membrane-associated replication complexes and support the concept that all are active in the synthesis of plus-strand RNAs.
Evolution of the Sabin type 1 poliovirus in humans: characterization of strains isolated from patients with vaccine-associated paralytic poliomyelitis
Authors: Georgescu M M , Balanant J , Macadam A , Otelea D , Combiescu M , Combiescu A A , Crainic R , Delpeyroux F
Published: January 2020

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and in rare cases cause vaccine-associated paralytic poliomyelitis (VAPP). Reversion of vaccine strains toward a pathogenic phenotype is probably one of the main causes of VAPP, a disease most frequently associated with type 3 and type 2 strains and more rarely with the type 1 (Sabin 1) strain. To identify the determinants and mechanisms of safety versus pathogenicity of the Sabin 1 strain, we characterized the genetic and phenotypic changes in six Sabin 1-derived viruses isolated from immunocompetent patients with VAPP. The genomes of these strains carried either few or numerous mutations from the original Sabin 1 genome. As assessed in transgenic mice carrying the human poliovirus receptor (PVR-Tg mice), all but one strain had lost the attenuated phenotype. Four strains presented only a moderate neurovirulent phenotype, probably due at least in part to reversions to the wild-type genotype, which were detected in the 5' noncoding region of the genome. The reversions found in most strains at nucleotide position 480, are known to be associated with an increase in neurovirulence. The construction and characterization of Sabin 1 mutants implicated a reversion at position 189, found in one strain, in the phenotypic change. The presence of 71 mutations in one neurovirulent strain suggests that a vaccine-derived strain can survive for a long time in humans. Surprisingly, none of the strains analyzed were as neurovirulent to PVR-Tg mice as was the wild-type parent of Sabin 1 (Mahoney) or a previously identified neurovirulent Sabin 1 mutant selected at a high temperature in cultured cells. Thus, in the human gut, the Sabin 1 strain does not necessarily evolve toward the genetic characteristics and high neuropathogenicity of its wild-type parent.
Neonatal DNA immunization with a plasmid encoding an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge
Authors: Hassett D E , Zhang J , Whitton J L
Published: January 2020

Conventional vaccines are remarkably effective in adults but are much less successful in the very young, who are less able to initiate a mature immune response and who may carry maternal antibodies which inactivate standard vaccines. We set out to determine whether DNA immunization might circumvent these problems. We have previously shown that intramuscular injection of plasmid DNA encoding the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) is capable of inducing immune responses and protecting 50% of adult mice against lethal and sublethal challenge with LCMV. Here we demonstrate that mouse pups injected with the same plasmid hours or days after birth produce major histocompatibility complex-restricted, NP-specific cytotoxic T lymphocytes (CTL) that persist into adulthood; 48% of vaccinated pups responded to subsequent sublethal viral challenge by the accelerated production of anti-NP LCMV-specific CTL, indicating that these animals had been successfully immunized by the plasmid DNA. In addition, these mice showed a >95% reduction in splenic viral titers 4 days postinfection compared to control mice, demonstrating a more rapid control of infection in vivo. Furthermore, pups born of and suckled on LCMV-immune dams (and therefore containing passively acquired anti-LCMV antibodies at the time of DNA inoculation) responded to the DNA vaccine in a similar manner, showing that maternally derived anti-LCMV antibodies do not significantly inhibit the generation of protective immune responses following DNA vaccination. These findings suggest that, at least in this model system, DNA immunization circumvents many of the problems associated with neonatal immunization.
A novel transcriptional element in circular DNA monomers of the duck hepatitis B virus
Authors: Beckel-Mitchener A , Summers J
Published: January 2020

We report the presence of two elements, pet and net, that are required for proper transcription of the duck hepatitis B virus (DHBV). These regions were previously identified by using plasmid clones of the virus in transient expression assays (M. Huang and J. Summers, J. Virol. 68:1564-1572, 1994). In this study, we further analyzed these regions by using in vitro-synthesized circular DHBV DNA monomers to mimic the authentic transcriptional template. We observed that pet was required for pregenome transcription from circular viral monomers, and in the absence of pet-dependent transcription, expression of the viral envelope genes was increased. We found that deletion of net in circularized DNA monomers led to the production of abnormally long transcripts due to a failure to form 3' ends during transcription. In addition, we report the presence of a net-like region in the mammalian hepadnavirus woodchuck hepatitis virus. These results are consistent with a model that net is a region involved in transcription termination and that in DHBV, pet is required for transcription complexes to read through this region during the first pass through net.
Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome
Authors: Surosky R T , Urabe M , Godwin S G , McQuiston S A , Kurtzman G J , Ozawa K , Natsoulis G
Published: January 2020

We have developed a system for site-specific DNA integration in human cells, mediated by the adeno-associated virus (AAV) Rep proteins. In its normal lysogenic cycle, AAV integrates at a site on human chromosome 19 termed AAVS1. We describe a rapid PCR assay for the detection of integration events at AAVS1 in whole populations of cells. Using this assay, we determined that the AAV Rep proteins, delivered in cis or trans, are required for integration at AAVS1. Only the large forms of the Rep protein, Rep78 and Rep68, promoted site-specific integration. The AAV inverted terminal repeats, present in cis, were not essential for integration at AAVS1, but in cells containing Rep, they increased the efficiency of integration. In the presence of the Rep proteins, the integration of a plasmid containing AAV inverted terminal repeats occurred at high frequency, such that clones containing the plasmid could be isolated without selection. In two of the five clones analyzed by fluorescence in situ hybridization, the plasmid DNA was integrated at AAVS1. In most of the clones, at least one copy of the entire plasmid was integrated in a tandem array. Detailed analysis of the integrated plasmid structure in one clone suggested a complex mechanism producing rearrangements of the flanking genomic DNA, similar to those observed with wild-type AAV.
CD46 expression does not overcome the intracellular block of measles virus replication in transgenic rats
Authors: Niewiesk S , Schneider-Schaulies J , Ohnimus H , Jassoy C , Schneider-Schaulies S , Diamond L , Logan J S , ter Meulen V
Published: January 2020

The study of measles pathogenesis and the testing of improved vaccine candidates is hampered by the lack of a small animal model which is susceptible to infection by the intranasal route. With the identification of CD46 as a measles virus (MV) receptor, it was feasible to generate transgenic rats to overcome this problem. Although there was widespread expression of CD46 in the transgenic Sprague-Dawley rats, no measles-like disease could be induced after various routes of infection. The expressed transgenic protein was functionally intact since it mediated MV fusion and was downregulated by contact with MV hemagglutinin. In vitro studies revealed that CD46-expressing rat fibroblasts take up MV but do not allow viral replication, which explains the nonpermissiveness of the transgenic rats for in vivo infection.
Influence of membrane anchoring and cytoplasmic domains on the fusogenic activity of vesicular stomatitis virus glycoprotein G
Authors: Odell D , Wanas E , Yan J , Ghosh H P
Published: January 2020

Chimeric proteins in which the transmembrane anchoring sequence (TM) or both the TM and the cytoplasmic tail (CT) of vesicular stomatitis virus glycoprotein G were replaced with corresponding domains of viral or cellular integral membrane proteins were used to examine the influence of these domains on acidic-pH-induced membrane fusion by G protein. The TM and CT of G were also replaced with the lipid anchor glycosylphosphatidylinositol. Hybrids containing foreign TM or TM and CT sequences were fusogenic at acidic pH but glycosylphosphatidylinositol-anchored G was nonfusogenic at acidic pH. The results suggest that the fusogenic activity of G protein requires membrane anchoring by a hydrophobic peptide sequence and the specific amino acid sequence of the TM has no influence on fusogenic activity.
Partial resistance to gD-mediated interference conferred by mutations affecting herpes simplex virus type 1 gC and gK
Authors: Pertel P E , Spear P G
Published: January 2020

Cells expressing herpes simplex virus (HSV) gD can be resistant to HSV entry as a result of gD-mediated interference. HSV strains differ in sensitivity to this interference, which blocks viral penetration but not binding. Previous studies have shown that mutations or variations in virion-associated gD can confer resistance to gD-mediated interference. Here we show that HSV-1 mutants selected for enhanced ability to bind and penetrate in the presence of inhibitory concentrations of heparin were partially resistant to gD-mediated interference. The resistance was largely due to the presence of two mutations: one in gC (the major heparin-binding glycoprotein) resulting in the absence of gC expression and the other in gK resulting in a syncytial phenotype. The results imply that heparin selected for mutants with altered postbinding requirements for entry. Resistance to gD-mediated interference conferred by mutations affecting gC and gK has not been previously described.
Competitive binding of viral E2 protein and mammalian core-binding factor to transcriptional control sequences of human papillomavirus type 8 and bovine papillomavirus type 1
Authors: Schmidt H M , Steger G , Pfister H
Published: January 2020

The promoter P7535 of human papillomavirus type 8 and the promoter P7185 of bovine papillomavirus type 1 are negatively regulated by viral E2 proteins via the promoter proximal binding sites P2 and BS1, respectively. Mutations of these E2 binding sites can reduce basal promoter activity. This suggests binding of a transcription-stimulating factor and may indicate that repression by E2 is due to competitive binding of viral and cellular proteins. A computer search revealed putative binding sites for core-binding factor (CBF; also referred to as PEA2, PEBP2, or AML), overlapping with P2 and BS1. Binding of recombinant CBF proteins to these sites was confirmed by band shift analysis. Competition of CBF and E2 protein for DNA binding was shown for both human papillomavirus type 8 and bovine papillomavirus type 1. The importance of CBF-E2 competition in E2-mediated repression could be demonstrated by comparing the E2 effect on P7185 activity in two cell lines containing different amounts of endogenous CBF. In cells with large amounts of CBF, E2 repressed P7185 wild-type constructs to the basal promoter activity of a mutant (50%) that could not bind this protein any more. In contrast, in a cell line containing small amounts of CBF, the promoter activities of constructs with wild-type and mutated CBF binding sites hardly differed and specific repression by E2 was not detectable.
Localization of the labile disulfide bond between SU and TM of the murine leukemia virus envelope protein complex to a highly conserved CWLC motif in SU that resembles the active-site sequence of thiol-disulfide exchange enzymes
Authors: Pinter A , Kopelman R , Li Z , Kayman S C , Sanders D A
Published: January 2020

Previous studies have indicated that the surface (SU) and transmembrane (TM) subunits of the envelope protein (Env) of murine leukemia viruses (MuLVs) are joined by a labile disulfide bond that can be stabilized by treatment of virions with thiol-specific reagents. In the present study this observation was extended to the Envs of additional classes of MuLV, and the cysteines of SU involved in this linkage were mapped by proteolytic fragmentation analyses to the CWLC sequence present at the beginning of the C-terminal domain of SU. This sequence is highly conserved across a broad range of distantly related retroviruses and resembles the CXXC motif present at the active site of thiol-disulfide exchange enzymes. A model is proposed in which rearrangements of the SU-TM intersubunit disulfide linkage, mediated by the CWLC sequence, play roles in the assembly and function of the Env complex.