bluuebunny/crossref_metadata_2025 huggingface dataset:
Antiviral immune responses in CTLA4 transgenic mice
Authors: Zimmermann C , Seiler P , Lane P , Zinkernagel R M
Published: January 2020

The role of B7 binding CD28 in the regulation of T- and B-cell responses against viral antigens was assessed in transgenic mice expressing soluble CTLA4-Hgamma1 (CTLA4-Ig tg mice) that blocks B7-CD28 interactions. The results indicate that transgenic soluble CTLA4 does not significantly alter cytotoxic T-cell responses against replicating lymphocytic choriomeningitis virus (LCMV) or vaccinia virus but drastically impairs the induction of cytotoxic T-cell responses against abortively replicating vesicular stomatitis virus (VSV). While the T-independent neutralizing immunoglobulin M (IgM) responses were within normal ranges, the switch to IgG was reduced 4- to 16-fold after immunization with abortively replicating VSV and more than 30-fold after immunization with an inert VSV glycoprotein antigen in transgenic mice. IgG antibody responses to LCMV, as detected by enzyme-linked immunosorbent assay and by neutralizing action, were reduced about 3- to 20-fold and more than 50-fold, respectively. These results suggest that responses in CTLA4-Ig tg mice are mounted according to their independence of T help. While immune responses to nonreplicating or poorly replicating antigens are in general most dependent on T help and B7-CD28 interactions, they are most impaired in CTLA4-Ig tg mice. The results of the present experiments also indicate that highly replicating viruses, because of greater quantities of available antigens and by inducing as-yet-undefined factors and/or cell surface changes, are capable of compensating for the decrease in T help caused by the blocking effects of soluble CTLA4.
Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1
Authors: Chappell J D , Gunn V L , Wetzel J D , Baer G S , Dermody T S
Published: January 2020

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.
Analysis of recombinant adeno-associated virus packaging and requirements for rep and cap gene products
Authors: Vincent K A , Piraino S T , Wadsworth S C
Published: January 2020

Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Because the gene transfer vector commonly retains only the AAV terminal repeats, propagation of recombinant AAV (rAAV) requires that the viral replication (Rep) and capsid (Cap) proteins be supplied in trans. In an effort to optimize the production of these vectors, a panel of helper plasmids was constructed to determine if expression of the rep and/or cap genes is a limiting factor for rAAV packaging. Expression of the Rep and Cap proteins was increased by replacing the endogenous AAV promoters, p5 and p40, with the Rous sarcoma virus (RSV) long terminal repeat (LTR) and the cytomegalovirus immediate-early promoter, respectively. Increased synthesis of the Cap proteins resulted in an approximately 10-fold increase in the yield of rAAV, indicating that production of capsid proteins is one limiting factor for rAAV packaging. Expression of the rep gene from the RSV LTR not only failed to increase the yield of rAAV but also prevented activation of p40 transcription with adenovirus infection, resulting in a reduced level of capsid protein synthesis.
Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax
Authors: Lemasson I , Robert-Hebmann V , Hamaia S , Duc Dodon M , Gazzolo L , Devaux C
Published: January 2020

To understand the mechanism of p56lck protein downregulation observed in human T cells infected by human T-cell leukemia virus type 1 (HTLV-1), we have investigated the ability of the 3' end of the HTLV-1 genome as well as that of the tax and rex genes to modulate p56lck protein expression and p56lck mRNA synthesis. By using Jurkat T cells stably transfected with constructs that expressed either the 3' end of the HTLV-1 genome (JK C11-pMTEX), the tax gene (JK52-Tax) or the rex gene (JK9-Rex), we found that the expression of p40tax (Tax) was sufficient to modulate p56lck protein expression. Similarly, we found that the expression of the mRNA which encoded p56lck was repressed in Jurkat T cells which expressed Tax. This downregulation was shown to be proportional to the amount of tax mRNA found in the transfected cells, as evidenced by experiments that used cells (JPX-9) stably transfected with a tax gene driven by a cadmium-inducible promoter. Furthermore, cadmium induction of Tax in JPX-9 cells transiently transfected with a construct containing the chloramphenicol acetyltransferase (CAT) gene under control of the lck distal promoter (lck DP-CAT) resulted in the downregulation of CAT gene expression. In contrast, cadmium induction of Tax in JPX-9 cells transiently transfected with a CAT construct driven by a lck DP with a deletion extending from position -259 to -253 (a sequence corresponding to a putative E-Box) did not modulate CAT gene expression, suggesting that the effect of Tax on p56lck is mediated through an E-Box binding protein.
Kaposi's sarcoma-associated herpesvirus encodes a functional cyclin
Authors: Li M , Lee H , Yoon D W , Albrecht J C , Fleckenstein B , Neipel F , Jung J U
Published: January 2020

Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) is consistently found in Kaposi's sarcoma lesions and in body-cavity-based lymphomas. A 17-kb KSHV lambda clone was obtained directly from a Kaposi's sarcoma lesion. DNA sequence analysis of this clone identified an open reading frame which has 32% amino acid identity and 53% similarity to the virus-encoded cyclin (v-cyclin) of herpesvirus saimiri (HVS) and 31% identity and 53% similarity to human cellular cyclin D2. This KSHV open reading frame was shown to encode a 29- to 30-kDa protein with the properties of a v-cyclin. KSHV v-cyclin protein was found to associate predominantly with cdk6, a cellular cyclin-dependent kinase known to interact with cellular type D cyclins and HVS v-cyclin. The KSHV v-cyclin was also found to associate weakly with cdk4. KSHV v-cyclin-cdk6 complexes strongly phosphorylated glutathione S-transferase-Rb fusion protein and histone H1 as substrates in vitro. Thus, KSHV v-cyclin resembles the v-cyclin of the T-lymphocyte-transforming HVS in its specificity for association with cdk6 and in its ability to strongly activate cdk6 protein kinase activity.
CCAAT displacement protein, a regulator of differentiation-specific gene expression, binds a negative regulatory element within the 5' end of the human papillomavirus type 6 long control region
Authors: Pattison S , Skalnik D G , Roman A
Published: January 2020

We have reported previously that a 636-bp fragment spanning the 5' two-thirds of the human papillomavirus type 6 (HPV6)-W50 long control region (LCR) functions as a transcriptional silencer (A. Farr, S. Pattison, B.-S. Youn, and A. Roman, J. Gen. Virol. 76:827-835, 1995). We have utilized nested deletion analyses to implicate a 66-bp sequence which appears to be critical for this activity. A comparison of the transcriptional regulatory activities of the LCRs of HPV6-W50 and HPV6b (which has a 94-bp deletion, resulting in the elimination of the 66-bp sequence) indicates that sequences within the 94-bp region negatively regulate the activity of the intact HPV6 LCR. Two sequence-specific DNA-protein interactions were visualized via electrophoretic mobility shift assays. One of the binding events is mediated by the transcriptional repressor CCAAT displacement protein (CDP), a factor which is active in undifferentiated cells but inactive in terminally differentiated cells. This conclusion is based on the following three lines of evidence: (i) a consensus CDP binding site oligonucleotide serves as a competitor in band shift assays, (ii) the band shift complex is not seen when a CDP-negative nuclear extract is used, and (iii) anti-CDP antiserum specifically inhibits the binding. These studies identify a DNA-protein interaction occurring within the 5' end of the LCR which may be important in maintaining the tight link between keratinocyte differentiation and HPV gene expression.
The role of ATF in regulating the human cytomegalovirus DNA polymerase (UL54) promoter during viral infection
Authors: Kerry J A , Priddy M A , Staley T L , Jones T R , Stenberg R M
Published: January 2020

Previous analysis of the human cytomegalovirus (HCMV) DNA polymerase (UL54) early gene promoter demonstrated that transcriptional activation of this gene is dependent upon the interaction of cellular transcription factors with viral transactivators (J. A. Kerry, M. A. Priddy, T. Y. Jervey, C. P. Kohler, T. L. Staley, C. D. Vanson, T. R. Jones, A. C. Iskenderian, D. G. Anders, and R. M. Stenberg, J. Virol. 70:373-382, 1996). A sequence element, IR1, was shown to be the primary regulatory element of this promoter in transient assays. However, assessment of this element in the context of the viral genome revealed IR1-independent activation at late times after infection. To extend these studies, we aim to identify additional sequence elements involved in the activation of the UL54 promoter. Our present studies demonstrate that the level of binding of proteins to the ATF site in the UL54 promoter is enhanced by viral infection. Furthermore this increase is sensitive to treatment with phosphonoacetic acid (PAA), a DNA synthesis inhibitor. These data suggest that the increase in the level of ATF binding activity is regulated, either directly or indirectly, by HCMV late gene expression. By using specific antibodies, we determined that ATF-1 was a major component of the proteins binding to the UL54 ATF site at late times. In addition, we have demonstrated direct binding of recombinant ATF-1 to the UL54 ATF site. To assess the biological significance of these events, a recombinant virus construct was generated that contained the UL54 promoter with a mutation in the ATF site regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene inserted between open reading frames US9 and US10. Analysis of this virus (RVATFmCAT) revealed that mutation of the ATF site does not alter the kinetics of UL54 promoter activation. However, levels of CAT mRNA and activity were reduced by 5- to 10-fold compared to those of the wild-type promoter at all stages of infection. These findings indicate that ATF-1 can regulate the levels of UL54 promoter activity at both early and late times. Furthermore, these results imply that HCMV can regulate the activity of cellular factors involved in early gene regulation.
Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus
Authors: Lane T E , Paoletti A D , Buchmeier M J
Published: January 2020

Intranasal inoculation of the neuroattenuated OBLV60 strain of mouse hepatitis virus results in infection of mitral neurons in the olfactory bulb, followed by spread along olfactory and limbic pathways to the brain. Immunocompetent BALB/c mice were able to clear virus by 11 days postinfection (p.i.). Gamma interferon (IFN-gamma) may play a role in clearance of OBLV60 from infected immunocompetent BALB/c mice through a nonlytic mechanism. Among the variety of immunomodulatory activities of IFN-gamma is the induction of expression of inducible nitric oxide synthase (iNOS), an enzyme responsible for the production of nitric oxide (NO). Studies were undertaken to investigate the role of IFN-gamma and NO in host defense and clearance of OBLV60 from the central nervous system (CNS). Exposure of OBLV60-infected OBL21a cells, a mouse neuronal cell line, to the NO-generating compound S-nitroso-L-acetyl penicillamine resulted in a significant decrease in viral replication, indicating that NO interfered with viral replication. Furthermore, infection of IFN-gamma knockout (GKO) mice and athymic nude mice with OBLV60 resulted in low-level expression of iNOS mRNA and protein in the brains compared to that of OBLV60-infected BALB/c mice. Nude mice were unable to clear virus and eventually died between days 11 and 14 p.i. (B. D. Pearce, M. V. Hobbs, T. S. McGraw, and M. J. Buchmeier, J. Virol. 68:5483-5495, 1994); however, GKO mice survived infection and cleared virus by day 18 p.i. These data suggest that IFN-gamma production in the olfactory bulb contributed to but may not be essential for clearance of OBLV60 from the brain. In addition, treatment of OBLV60-infected BALB/c mice with aminoguanidine, a selective inhibitor of iNOS activity, did not result in any increase in mortality, and the mice cleared the virus by 11 days p.i. These data suggest that although NO was able to block replication of virus in vitro, expression of iNOS with NO release in vivo did not appear to be the determinant factor in clearance of OBLV60 from CNS neurons.
Accumulation of defective viral genomes in peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected individuals
Authors: Sanchez G , Xu X , Chermann J C , Hirsch I
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) genomes present in peripheral blood mononuclear cells (PBMCs) of infected persons or in lymphocytes infected in vitro were studied by long-distance PCR (LD-PCR) using primers localized in the HIV-1 long terminal repeats. The full-length 9-kb DNA was the only LD-PCR product obtained in peripheral and cord blood lymphocytes from seronegative donors infected in vitro. However, a high proportion (27% to 66%) of distinct populations of extensively deleted HIV-1 genomes of variable size was detected in PBMCs of 15 of 16 HIV-1-infected persons. Physical mapping of defective genomes showed that the frequency of deletions is proportional to their proximity to the central part of HIV-1 genome, which is consistent with a deletion mechanism involving a single polymerase jump during reverse transcription. Sequencing of deletion junctions revealed the presence of short direct repeats of three or four nucleotides. The number of defective HIV-1 genomes decreased after in vitro activation of PBMCs. Persistence of full-length and deleted genomes in in vitro activated PBMCs correlated with isolation of an infectious virus. Our results represent the first quantitative assessment of intragenomic rearrangements in HIV-1 genomes in PBMCs of infected persons and demonstrate that, in contrast to in vitro infection, defective genomes accumulate in PBMCs of infected persons.
Human CD4+ T-cell response to hepatitis delta virus: identification of multiple epitopes and characterization of T-helper cytokine profiles
Authors: Nisini R , Paroli M , Accapezzato D , Bonino F , Rosina F , Santantonio T , Sallusto F , Amoroso A , Houghton M , Barnaba V
Published: January 2020

The T-cell-mediated immune response plays a crucial role in defense against hepatotropic viruses as well as in the pathogenesis of viral chronic hepatitides. However, very little is known about the role of specific T cells during hepatitis delta virus (HDV) infection in humans. In this study, the T-cell response to HDV in chronic hepatitis B virus (HBV) carriers with HDV superinfection was investigated at different levels. Analysis of peripheral blood mononuclear cell (PBMC) proliferation in response to a recombinant form of large hepatitis delta antigen (HDAg) revealed that 8 of 30 patients studied (27%) specifically responded to HDAg. By employing synthetic peptides spanning the entire HDAg sequence, we found that T-cell recognition was directed against different antigenic determinants, with patient-to-patient variation in the pattern of response to peptides. Interestingly, all responders had signs of inactive HDV-induced disease, while none of the patients with active disease and none of the control subjects showed any significant proliferation. More accurate information about the specific T-cell response was obtained at the clonal level. A panel of HDAg-specific CD4+ T-cell clones from three HDV-infected individuals and fine-specificity analysis revealed that the clones tested individually recognized four epitopes corresponding to amino acids (aa) 26 to 41, 50 to 65, 66 to 81, or 106 to 121 of HDAg sequence. The study of human leukocyte antigen (HLA) restriction revealed that peptides 50 to 65 and 106 to 121 were presented to specific T cells in association with multiple class II molecules. In addition, peptide 26 to 41 was efficiently generated after processing of HDAg through the endogenous processing pathway. Cytokine secretion analysis showed that all the CD4+ T-cell clones assayed were able to produce high levels of gamma interferon (IFN-gamma), belonging either to T helper-1 (Th1) or Th0 subsets and that some of them were cytotoxic in a specific assay. This study provides the first evidence that detection of a specific T-cell response to HDAg in the peripheral blood of individuals with hepatitis delta is related to the decrease of HDV-induced disease activity. The HDAg epitopes identified here and particularly those recognized by CD4+ T cells in association with multiple major histocompatibility complex class II molecules may be potentially exploited for the preparation of a vaccine for prophylaxis and therapy of HDV infection.
In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs
Authors: Houser-Scott F , Ansel-McKinney P , Cai J M , Gehrke L
Published: January 2020

The coat proteins of alfalfa mosaic virus (AMV) and the related ilarviruses bind specifically to the 3' untranslated regions of the viral RNAs, which contain conserved repeats of the tetranucleotide sequence AUGC. The purpose of this study was to develop a more detailed understanding of RNA sequence and/or structural determinants required for coat protein binding by characterizing the role of the AUGC repeats. Starting with a complex pool of 39-nucleotide RNA molecules containing random substitutions in the AUGC repeats, in vitro genetic selection was used to identify RNAs that bound coat protein. After six iterative rounds of selection, amplification, and reselection, 25% of the RNAs selected from the randomized pool were wild type; that is, they contained all four AUGC sequences. Among the 31 clones analyzed, AUGC was clearly the preferred selected sequence at the four repeats, but some nucleotide sequence variability was observed at AUGC(865-868) if the other three AUGC repeats were present. Variant RNAs that bound coat protein with affinities equal to or greater than that of the wild-type molecule were not selected. To extend the in vitro selection results, RNAs containing specific nucleotide substitutions were transcribed in vitro and tested in coat protein and peptide binding assays. The data strongly suggest that the AUGC repeats provide sequence-specific determinants and contribute to a structural platform for specific coat protein binding. Coat protein may function in maintaining the 3' ends of the genomic RNAs during replication by stabilizing an RNA structure that defines the 3' terminus as the initiation site for minus-strand synthesis.
Failure to transmit disease from gray tremor mutant mice
Authors: Carlson G A , Banks S , Lund D , Reichert C , Groth D , Torchia M , Dearmond S J , Prusiner S B
Published: January 2020

Mice homozygous for mutant alleles at the gray tremor (gt) locus develop a marked non-intention tremor beginning at 8 days of age. Most homozygous mice die by 3 months. Homozygotes exhibit intense vacuolation of the central nervous system gray matter and vacuolation and hypomyelination of some white matter tracts. Based on neuropathological similarities with scrapie, other investigators inoculated wild-type mice with gray tremor brain homogenates to test the hypothesis of transmissibility. Published reports indicated that spongiform encephalopathy (R. L. Sidman, H. C. Kinney, and H. O. Sweet, Proc. Natl. Acad. Sci. USA 82:253-257, 1985) and disease, including hind limb paralysis in NFS mice (P. M. Hoffman, R. G. Rohwer, C. MacAuley, J. A. Bilello, J. W. Hartley, and H. C. Morse III, Proc. Natl. Acad. Sci. USA 84:3866-3870, 1987), were transmitted by inoculation of gt/gt brain homogenates. In our hands, however, no NFS/NCr animals inoculated intracerebrally with gt/gt or +/+ brain preparations showed any signs of disease or pathological changes in the brain. Positive transmission by other investigators may reflect the microbiological status of their donor or recipient mice.
Induction of degenerative brain lesions after adoptive transfer of brain lymphocytes from Borna disease virus-infected rats: presence of CD8+ T cells and perforin mRNA
Authors: Sobbe M , Bilzer T , Gommel S , Nöske K , Planz O , Stitz L
Published: January 2020

Lymphocytes were isolated from the brains of Borna disease virus-infected donor Lewis rats at various time points after infection. Cell populations were characterized by cytofluorometry, with special emphasis on CD4+ and CD8+ cells. Testing of isolated lymphocytes revealed major histocompatibility complex class I-restricted cytotoxic activity. Reverse transcription-PCR analyses of brain homogenates of infected donors revealed the presence of CD8 mRNA after day 11 of infection and of perforin mRNA between days 13 and 25 after infection. Adoptive transfers of lymphocytes isolated from the brain at days 13 and 21 resulted in severe neurological symptoms, resembling experimental Borna disease. The onset of disease was dependent on the cell numbers transferred and was clearly related to the appearance of T cells in the brain. CD8+ T cells were found in the parenchyma, whereas CD4+ T cells were found predominantly in perivascular locations. A disseminated lymphocytic infiltration in the parenchyma was accompanied by severe morphological alterations, including significant necrosis of neurons. Furthermore, a prominent spongiform-like degeneration was observed; this increased over time and finally resulted in severe cortical brain atrophy. Lymphocytes obtained during the beginning chronic phase of experimental Borna disease in rats had no significant cytolytic capacity in vitro and were also not able to induce neurological symptoms typical of Borna disease after adoptive transfer. The data presented here show for the first time that lymphocytes isolated from the site of the inflammatory lesions, namely, the brains of diseased rats, induce the immunopathological reaction and cause Borna disease. After transfer, the pathological alterations induced in the recipients exactly reflect those observed during experimentally induced Borna disease in rats, including necrosis of neurons and glial cells and gross degeneration resulting in cortical brain atrophy. Evidence that the immunopathology of Borna disease is closely related to the presence of CD8+ T cells in the brain parenchyma is provided.
Effect of the E4 region on the persistence of transgene expression from adenovirus vectors
Authors: Armentano D , Zabner J , Sacks C , Sookdeo C C , Smith M P , St George J A , Wadsworth S C , Smith A E , Gregory R J
Published: January 2020

The utility of adenovirus vectors for gene therapy is limited by the transience of expression that has been observed in various in vivo models. Immunological responses to viral targets can eliminate transduced cells and cause the loss of transgene expression. We previously described the characterization of an E4 modified adenovirus, Ad2E4ORF6, which is replication defective in cotton rats. We reasoned that gene transfer vectors based on Ad2E4ORF6 would have a reduced potential for viral gene expression in vivo which might be beneficial for achieving persistence of transgene expression. E1 replacement vectors expressing the cystic fibrosis transmembrane regulator or beta-galactosidase were constructed as series of vectors that differed with respect to the E4 region. Vectors containing a wild-type E4 region, E4 open reading frame 6, or a complete E4 deletion were compared in the lungs of BALB/c mice for persistence of expression. Results obtained with nude mice indicate that nonimmunological factors have a major influence on the longevity of transgene expression. Expression was transient from the E1a promoter with all vectors but persisted from the cytomegalovirus promoter only with a vector containing a wild-type E4 region. Transience of expression did not correlate with the disappearance of vector DNA, suggesting that promoter down-regulation may be involved. Coinfection studies indicate an E4 product(s) could be supplied in trans to allow persistent expression from the cytomegalovirus promoter. In summary, the choice of promoter is important for achieving persistence of expression; in addition, some promoters are highly influenced by the context of the vector backbone.
Phenotypically Vif- human immunodeficiency virus type 1 is produced by chronically infected restrictive cells
Authors: Bouyac M , Rey F , Nascimbeni M , Courcoul M , Sire J , Blanc D , Clavel F , Vigne R , Spire B
Published: January 2020

The permissivity of CD4+ transformed T cells for the replication of human immunodeficiency virus type 1 (HIV-1) vif mutants varies widely between different cell lines. Mutant vif-negative viruses propagate normally in permissive CD4+ cell lines but are unable to establish a productive infection in restrictive cell lines such as H9. As a consequence, elucidation of the function of Vif has been considerably hampered by the inherent difficulty in obtaining a stable source of authentically replication-defective vif-negative viral particles produced by restrictive cells. vif-negative, vpr-negative HIV-1 strain NDK stock, produced by the permissive SupT1 cell line, was used to infect restrictive H9 cells. By using a high multiplicity, infection of H9 cells was achieved, leading to persistent production of viral particles displaying a dramatically reduced infectious virus titer when measured in a single-cycle infectivity assay. Although these viral particles were unable to further propagate in H9 cells, they could replicate normally in CEM and SupT1 cells. Comparison of unprocessed and processed Gag proteins in the persistently produced vif-negative viral particles revealed no defect in the processing of polypeptide precursors, with no inversion of the Pr55gag/p24 ratio. In addition, there was no defect in Env incorporation for the vif-negative viral particles. Despite their apparently normal protein content, these particles were morphologically abnormal when examined by transmission electron microscopy, displaying a previously described abnormally condensed nucleoid. Chronically infected restrictive cell lines producing stable levels of phenotypically vif-negative HIV-1 particles could prove particularly useful in further studies on the function of Vif in the virus life cycle.
Molecular determinants for cellular uptake of Tat protein of human immunodeficiency virus type 1 in brain cells
Authors: Ma M , Nath A
Published: January 2020

We measured the cellular uptake of 125I-labeled full-length Tat (amino acids 1 to 86) (125I-Tat(1-86)) and 125I-Tat(1-72) (first exon) in human fetal astrocytes, neuroblastoma cells, and human fetal neurons and demonstrated that the uptake of 125I-Tat(1-72) without the second exon was much lower than that of 125I-Tat(1-86) (P < 0.01). This suggests an important role for the C-terminal region of Tat for its cellular uptake. 125I-Tat uptake could be inhibited by dextran sulfate and competitively inhibited by unlabeled Tat but not by overlapping 15-mer peptides, suggesting that Tat internalization is charge and conformationally dependent. Interestingly, one of 15-mer peptides, Tat(28-42), greatly enhanced 125I-Tat uptake. These findings are important for understanding the neuropathogenesis of human immunodeficiency virus type 1 infection and in the potential application of Tat for drug delivery to cells.
Host-specific driving force in human immunodeficiency virus type 1 evolution in vivo
Authors: Zhang L , Diaz R S , Ho D D , Mosley J W , Busch M P , Mayer A
Published: January 2020

To investigate the process of human immunodeficiency virus type 1 (HIV-1) evolution in vivo, a total of 179 HIV-1 V3 sequences derived from cell-free plasma were determined from serial samples in three epidemiologically linked individuals (one infected blood donor and two transfusion recipients) over a maximum period of 8 years. A systematic analysis of pairwise comparisons of intrapatient sequences, both within and between each sample time point, revealed a preponderance and accumulation of nonsynonymous rather than synonymous substitutions in the V3 loop and flanking regions as they diverged over time. This strongly argues for the dominant role that positive selection for amino acid change plays in governing the pattern and process of HIV-1 env V3 evolution in vivo and nullifies hypotheses of purely neutral or mutation-driven evolution or completely chance events. In addition, different rates of evolution of HIV-1 were observed in these three different individuals infected with the same viral strain, suggesting that the degree of positive pressure for HIV-1 amino acid change is host dependent. Finally, the observed similar rate of accumulation in divergence within and between infected individuals suggests that the process of genetic divergence in the HIV epidemic proceeds regardless of host-to-host transmission events, i.e., that transmission does not reset the evolutionary clock.
Inhibition of recombinant human immunodeficiency virus type 1 replication by a site-specific recombinase
Authors: Flowers C C , Woffendin C , Petryniak J , Yang S , Nabel G J
Published: January 2020

Current molecular genetic strategies to inhibit productive human immunodeficiency virus type 1 (HIV-1) replication have involved the generation of gene products which provide intracellular inhibition of essential virally encoded proteins or RNA structures. A molecular strategy to excise proviral DNA from HIV-1-infected cells and render these cells virus free would provide an attractive direct antiviral strategy, providing a mechanism to remove viral genes from infected cells. The potential of such a molecular genetic intervention was examined by using the Cre-loxP recombination system. A recombinant HIV-1 clone, designated HIV(lox), that contains loxP within a nonessential U3 region of the long terminal repeats was synthesized. The loxP motif was maintained during replication of HIV(lox) in CEM cells, as demonstrated by reverse transcriptase PCR analyses of genomic RNA isolated from virions. Two different types of HIV-1-permissive cells, CEM cells and 293 cells expressing the CD4 glycoprotein, were transformed with a Cre expression vector which was shown to encode Cre DNA binding and recombinase activities. HIV(lox) infection of CEM or CD4+ 293 cells expressing Cre resulted in a substantial reduction in virus replication compared to control cells, and evidence for the presence of the expected excision product was found. Site-specific excision of HIV-1 can therefore be achieved by using this model system with acute infection. These studies represent one step toward the development of a novel antiviral strategy for the treatment of AIDS.
RNA-RNA recombination in Sindbis virus: roles of the 3' conserved motif, poly(A) tail, and nonviral sequences of template RNAs in polymerase recognition and template switching
Authors: Hill K R , Hajjou M , Hu J Y , Raju R
Published: January 2020

Sindbis virus (SIN), a mosquito-transmitted animal RNA virus, carries a 11.7-kb positive-sense RNA genome which is capped and polyadenylated. We recently reported that the SIN RNA-dependent RNA polymerase (RdRp) could initiate negative-strand RNA synthesis from a 0.3-kb 3'-coterminal SIN RNA fragment and undergo template switching in vivo (M. Hajjou, K. R. Hill, S. V. Subramaniam, J. Y. Hu, and R. Raju, J. Virol. 70:5153-5164, 1996). To identify and characterize the viral and nonviral sequences which regulate SIN RNA synthesis and recombination, a series of SIN RNAs carrying altered 3' ends were tested for the ability to produce infectious virus or to support recombination in BHK cells. The major findings of this report are as follows: (i) the 3'-terminal 20-nucleotides (nt) sequence along with the abutting poly(A) tail of the SIN genome fully supports negative-strand synthesis, genome replication, and template switching; (ii) a full-length SIN RNA carrying the 3'-terminal 24 nt but lacking the poly(A) tail is noninfectious; (iii) SIN RNAs which carry 3' 64 nt or more without the poly(A) tail are infectious and regain their poly(A) tail in vivo; (iv) donor templates lacking the poly(A) tail do not support template switching; (v) full-length SIN RNAs lacking the poly(A) tail but carrying 3' nonviral extensions, although debilitated to begin with, evolve into rapidly growing poly(A)-carrying mutants; (vi) poly(A) or poly(U) motifs positioned internally within the acceptor templates, in the absence of other promoter elements within the vicinity, do not induce the jumping polymerase to reinitiate at these sites; and (vii) the junction site selection on donor templates occurs independently of the sequences around the acceptor sites. In addition to furthering our understanding of RNA recombination, these studies give interesting clues as to how the alphavirus polymerase interacts with its 3' promoter elements of genomic RNA and nonreplicative RNAs. This is the first report that an in vitro-synthesized alphavirus RNA lacking a poly(A) tail can initiate infection and produce 3' polyadenylated viral genome in vivo.
Pseudorabies virus recombinants expressing functional virulence determinants gE and gI from bovine herpesvirus 1.1
Authors: Knapp A C , Enquist L W
Published: January 2020

In the Alphaherpesvirinae subfamily, the gE and gI genes are conserved and encode membrane glycoproteins required for efficient pathogenesis (virulence). The molecular mechanism(s) responsible is not well understood, but the existence of similar phenotypes of gE and gI mutations in diverse Alphaherpesvirinae implies conservation of function(s). In this report, we describe construction of pseudorabies virus (PRV) recombinants that efficiently express the bovine herpesvirus 1 (BHV-1) membrane proteins gI and gE at the PRV gG locus. Each BHV-1 gene was cloned in a PRV mutant lacking both the PRV gI and gE coding sequences. All recombinant viruses expressed the BHV-1 proteins at levels similar to or greater than that observed after infection with parental BHV-1, and there were no observable differences in processing or ability to form gE-gI oligomers. The important observation resulting from this report is that the BHV-1 gE and gI proteins functioned together to complement the virulence defect of PRV lacking its own gE and gI genes in a rodent model, despite being derived from a highly restricted host range virus with a different pathogenic profile.
Sindbis virus replicons and Sindbis virus: assembly of chimeras and of particles deficient in virus RNA
Authors: Frolov I , Frolova E , Schlesinger S
Published: January 2020

Alphaviruses are a well-characterized group of positive-strand RNA viruses. The identification of cis-acting elements in their genomes and their replication strategy have made them useful as vectors for the expression of heterologous genes. In infected cells, the nonstructural proteins, required for replication and transcription of the viral genes, are translated from the genomic RNA; the structural proteins, the capsid protein that interacts with the RNA to form the nucleocapsid and the proteins embedded in the lipid envelope, are translated from a subgenomic mRNA and can be replaced by heterologous genes. Such modified genomes are self-replicating (replicons); they can be introduced into the cells by transfection and can also be packaged into extracellular particles with defective helper (DH) RNAs. The particular DH RNA determines how well it is replicated and to what extent it is packaged. One potential complication of this system has been that recombination between the replicon genome and the DH RNA may occur. The studies described here were designed to prevent recombination by expressing the capsid protein from one DH RNA and the virus membrane proteins from a second helper RNA. Recombination to yield a nonsegmented infectious virus genome would then require several independent crossover events. There is a translational enhancer located downstream of the initiating AUG in the RNA of the capsid gene that had to be conserved in the second helper to achieve high-level expression of the viral glycoproteins. For this reason, we modified the capsid protein gene in two ways: the first was to use the capsid protein gene from a different alphavirus, Ross River virus, and the second was to make deletions in that gene to maintain the translational enhancer in the RNA but to eliminate the positively charged region in the protein that should be essential for the specific and nonspecific interactions with RNA. Transfections with replicon RNA and the deleted chimeric DH RNA as the only helper resulted in the high-level production of particles that were almost completely devoid of RNA. The inclusion of a helper expressing an intact Sindbis virus capsid protein gene led to the production of high levels of packaged replicons. Recombinants were not detected even after several undiluted passages.
Orally administered microencapsulated reovirus can bypass suckled, neutralizing maternal antibody that inhibits active immunization of neonates
Authors: Periwal S B , Speaker T J , Cebra J J
Published: January 2020

Purified reovirus serotype 1, encapsulated in biodegradable aqueous microcapsules, was found to bypass maternal antibody passively transferred by suckling to neonates. Genetically identical, immunocompetent F1 scid/+ mice were generated by the reciprocal crosses of C.B17 scid/scid and normal congenic +/+ adult mice. The immunocompetent +/+ dams were either orally infected with reovirus prior to mating or not. Thus, these immunocompetent F1 pups developed either in the absence or in presence of passively transferred maternal immunity. The F1 mice were orally immunized on day 10 with either live virus, microencapsulated reovirus, or empty microcapsules plus live virus. The immune responses were assessed in the neonatal gut-associated lymphoid tissues (GALT). Examination of reovirus specific immunoglobulin A in the serum and GALT, taken on days 7, 14, and 21 postimmunization, clearly demonstrated that microencapsulated reovirus could bypass the normal effect of maternal antibodies, passively acquired by suckling, to inhibit active priming of neonates by oral route. These observations seem relevant to the development of efficacious oral vaccines that also allow passive, protective immunity via suckled maternal antibodies while permitting active oral immunization of neonates.
Characterization of synthetic foot-and-mouth disease virus provirions separates acid-mediated disassembly from infectivity
Authors: Knipe T , Rieder E , Baxt B , Ward G , Mason P W
Published: January 2020

One of the final steps in the maturation of foot-and-mouth disease virus (FMDV) is cleavage of the VP0 protein to produce VP4 and VP2. The mechanism of this cleavage is unknown, but it is thought to function in stabilizing the virus particle and priming it for infecting cells. To investigate the cleavage process and to understand its role in virion maturation, we engineered synthetic FMDV RNAs with mutations at Ala-85 (A85) and Asp-86 (D86) of VP0, which border the cleavage site. BHK cells transfected with synthetic RNAs containing substitutions at position 85 (A85N or A85H) or at position 86 (D86N) yielded particles indistinguishable from wild-type (WT) virus in sedimentation and electrophoretic profiles. Viruses derived from these transfected cells were infectious and maintained their mutant sequences upon passage. However, BHK cells transfected with synthetic RNAs encoding Phe and Lys at these positions (A85F/D86K) or a Cys at position 86 (D86C) produced noninfectious provirions with uncleaved VP0 molecules. Despite their lack of infectivity, the A85F/D86K provirions displayed cell binding and acid sensitivity similar to those of WT virus. However, acid breakdown products of the A85F/D86K provirions differed in hydrophobicity from the comparable WT virion products, which lack VP4. Taken together, these studies are consistent with a role for soluble VP4 molecules in release of the viral genome from the endosomal compartment of susceptible cells.
Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system
Authors: Gillock E T , Rottinghaus S , Chang D , Cai X , Smiley S A , An K , Consigli R A
Published: January 2020

Using the p2Bac dual multiple cloning site transfer vector, the polyomavirus major capsid protein gene VP1 was cloned for expression in the baculovirus-insect cell expression system. The 5-day-infected cellular lysate from this recombinant preparation was purified by cesium chloride density gradient centrifugation. Capsid-like particles were observed in the resulting preparation. The purified particle preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to have accurately expressed the polyomavirus VP1 protein as cloned. It was found that the preparation revealed the presence of host histones in the stained gels, which is indicative of DNA packaging. To determine if cellular DNA was being packaged in the particles, Sf9 insect cells were prelabeled with [3H] thymidine. The label was removed, and the cells were subsequently infected with a recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) carrying the polyomavirus VP1 gene. Upon purification through three cesium chloride gradients and DNase I treatment, capsid-like particles, containing [3H]thymidine-labeled DNA, were isolated which were found to coincide with hemagglutination activity. Studies have indicated that the AcMNPV appears to have the ability to fragment Sf9 cellular DNA. When infected with the recombinant AcMNPV carrying the VP1 gene of polyomavirus, these host DNA fragments are being packaged by the VPI major capsid protein; further, these DNA fragments have been shown to be approximately 5 kb in size, which corresponds to the size of the native polyomavirus genome. These studies demonstrate that the recombinant polyomavirus VP1 protein has the ability to package DNA in the absence of the minor structural proteins VP2 and VP3 and independently of the polyomavirus T antigens.
Transcomplementation of nucleotide priming and reverse transcription between independently expressed TP and RT domains of the hepatitis B virus reverse transcriptase
Authors: Lanford R E , Notvall L , Lee H , Beames B
Published: January 2020

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of the first deoxyribonucleotide to the polymerase polypeptide. We recently expressed human hepatitis B virus (HBV) reverse transcriptase (pol) in insect cells by using the recombinant baculovirus system. The purified protein is active in nucleotide priming and reverse transcription reactions. In this report, we demonstrate that the tyrosine residue at amino acid number 63 within the TP (terminal protein) domain of the polymerase is the site of covalent linkage of the first nucleotide of minus-strand DNA. Analysis of pol polypeptides with mutations in the TP and RT (reverse transcriptase) domains indicated that both domains were required for in vitro nucleotide priming activity. Polymerase proteins with mutations in the TP and RT domains were not capable of complementing each other in the nucleotide priming reaction, suggesting that transcomplementation between full-length polypeptides was not possible. However, when the TP and RT domains were expressed as separate polypeptides, they formed a highly stable complex that was active in nucleotide priming and reverse transcription. The presence of an epsilon stem-loop dramatically increased the nucleotide priming activity in transcomplementation assays, even though full-length pol displayed similar activities in the absence and presence of epsilon. These data raise the possibility that in the transcomplementation assay, epsilon may play a role in the formation of a functional complex between TP and RT, rather than being required only as the template for nucleotide priming. The results indicate that using the baculovirus system, it is possible to dissect the protein-protein and protein-RNA interactions required for HBV genome replication.
Chemical barriers to human immunodeficiency virus type 1 (HIV-1) infection: retrovirucidal activity of UC781, a thiocarboxanilide nonnucleoside inhibitor of HIV-1 reverse transcriptase
Authors: Borkow G , Barnard J , Nguyen T M , Belmonte A , Wainberg M A , Parniak M A
Published: January 2020

UC781, a thiocarboxanilide nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), inhibited RT DNA polymerase activity in vitro with marked potency. Significant inhibition was noted at a 1:1 molar ratio of UC871 to RT, characteristic of a tight-binding inhibitor. Infectivity of the HIV-1(IIIB) laboratory strain was eliminated in a concentration-dependent manner following short exposure of isolated virion particles to UC781. Neither nevirapine nor certain other carboxanilide nonnucleoside inhibitors were effective in this manner. Endogenous reverse transcription in UC781-treated virus particles was markedly reduced. Treatment of chronically HIV-1-infected H9 cells with UC781 did not alter virus production, but the infectivity of the virus produced by the cells during drug exposure was markedly reduced. Moreover, the infectivity of nascent virus produced by the UC781-treated H9 cells after removal of exogenous drug was dramatically attenuated. Similarly, pretreatment of peripheral blood lymphocytes isolated from HIV-infected patients abolished the infectivity of virus produced by these cells after removal of exogenous drug, as measured by coculture experiments with uninfected cord blood mononuclear cells, indicating the utility of UC781 against a variety of clinical HIV samples. Importantly, preincubation of uninfected MT2 cells with UC781 rendered these cells refractory to subsequent HIV infection in the absence of extracellular drug, an effect that persisted for several days following removal of exogenous drug. These unique properties of UC781 indicate that this nonnucleoside inhibitor may have considerable promise for use in retrovirucidal formulations to minimize the spread of HIV from infected to noninfected individuals.