bluuebunny/crossref_metadata_2025 huggingface dataset:
A vaccinia virus-vectored Hantaan virus vaccine protects hamsters from challenge with Hantaan and Seoul viruses but not Puumala virus
Authors: Chu Y K , Jennings G B , Schmaljohn C S
Published: January 2020

To investigate the ability of a vaccinia virus-vectored vaccine expressing the M and the S segments of Hantaan (HTN) virus (C. S. Schmaljohn, S. E. Hasty, and J. M. Dalrymple, Vaccine 10:10-13, 1992) to elicit a protective immune response against other hantaviruses, we vaccinated hamsters with the recombinant vaccine and challenged them with HTN, Seoul (SEO), or Puumala (PUU) virus. Neutralizing antibodies to HTN virus were found in all vaccinated hamsters both before and after challenge. Neutralizing antibody titers to SEO virus were present at low levels or were undetectable after two immunizations with the vaccine but were positive in all vaccinated hamsters after challenge with SEO virus and were also positive in control animals that were not challenged. Neutralizing antibodies to PUU virus were observed only in hamsters previously challenged with PUU virus. To assay for virus in the blood and tissues of the hamsters, we developed a nested reverse transcriptase (RT)-PCR with cross-reactive outer primers and serotype-specific inner primers. The RT-PCR specifically detected as little as 1 PFU of virus in serum containing high-titer neutralizing antibodies and was more sensitive than immunofluorescent antibody staining for detecting virus in lung and kidney specimens of infected hamsters. By using the RT-PCR, we found that vaccinated hamsters, challenged with HTN or SEO virus, neither were viremic nor had evidence of virus in their lungs or kidneys. In contrast, vaccinated hamsters challenged with PUU virus were viremic and had PUU virus-specific nucleic acid in their organs.
Rapid induction of apoptosis by cell-to-cell transmission of human immunodeficiency virus type 1
Authors: Maldarelli F , Sato H , Berthold E , Orenstein J , Martin M A
Published: January 2020

The kinetics of human immunodeficiency virus type 1 (HIV-1)-induced cell death were investigated in cell-to-cell and cell-free models of virus transmission. Cocultivation of HIV-1 chronically infected H9 donor cells with uninfected H9 recipient cells resulted in rapid induction of programmed cell death. Within 8 h, apoptotic chromatin condensation was identified by histologic staining. In addition, many single cells with apoptotic nuclei were observed, indicating that stable cell fusion was not a requirement for apoptosis to occur. By 12 to 18 h of coculture, a DNA fragmentation ladder characteristic of apoptosis was detected by agarose gel electrophoresis. Quantitation of apoptosis by measurement of nuclear DNA content revealed that at least 20 to 30% of the nuclei were undergoing apoptosis by 24 h after cocultivation. The appearance of condensed nuclei and fragmented DNA occurred as HIV reverse transcription was completed, and it was not inhibited by zidovudine, suggesting that induction of apoptosis did not require new HIV replication. Soluble CD4 inhibited apoptosis, demonstrating that Env-CD4 interactions were required for apoptosis. In contrast to that in cell-to-cell transmission, apoptosis in cell-free HIV infections was markedly inefficient and was not observed until 70 to 90 h after infections were initiated. These findings indicate that HIV-1 induction of programmed destruction of the nucleus is initiated at the time of cell-cell cocultivation by a mechanism which requires CD4-Env interactions but not new HIV replication.
Vesicular stomatitis virus infection of the central nervous system activates both innate and acquired immunity
Authors: Bi Z , Barna M , Komatsu T , Reiss C S
Published: January 2020

Vesicular stomatitis virus (VSV) causes acute infection of the central nervous system (CNS) when intranasally applied. We have examined cellular inflammatory changes in the CNS following VSV infection. As early as 1 day postinfection (p.i.), astrocytes were activated in the olfactory bulb (OB). This was followed by activation of microglia, first observed in the OB at day 3 p.i. Expression of inducible nitric oxide synthase was observed in activated microglia in the OB at day 3 p.i., and increased inducible nitric oxide synthase expression coincided with decreased virus titers in tissue homogenates. Expression of major histocompatibility complex (MHC) class I molecules on astrocytes and microglial, endothelial, and ependymal cells was also rapidly induced and followed by induced expression of MHC class II molecules on astrocytes and microglial and endothelial cells. Consistent with the pattern of viral dissemination, MHC molecules were expressed temporally from the rostral-to-caudal direction. Infiltration of CD8+ cells was observed as early as 1 day p.i. in the OB. CD4+ cells were detected in the OB at day 4 p.i. Increasing T-cell infiltration coincided with decreased virus titers. In contrast, B-cell infiltration of the CNS was not detected until day 14 p.i., after the virus was cleared and mice were showing behavioral signs of recovery. Breakdown of the blood-brain barrier was detected beginning at day 6 p.i., was most severe at day 8 p.i., and was followed by full recovery. Collectively, these data show that both innate immunity (production of nitric oxide) and acquired immunity (expression of MHC molecules and T-cell infiltration) are activated following VSV infection in the CNS.
Membrane binding and endoplasmic reticulum retention sequences of rotavirus VP7 are distinct: role of carboxy-terminal and other residues in membrane binding
Authors: Clarke M L , Lockett L J , Both G W
Published: January 2020

The sequences responsible for binding rotavirus glycoprotein VP7 to the membrane of the endoplasmic reticulum (ER) have not been identified. Here we show that the sequences which promote membrane binding in vitro are distinct from the N-terminal sequences which promote retention of VP7 in the ER in vivo. The role of the C-terminal region in membrane binding was also examined by using truncation mutants. Membrane binding in vitro was reduced but not abolished by removing up to 102 residues from the C terminus. The data suggest that the last 36 residues of VP7 may be present in the membrane or translocation pore, possibly with the C terminus protruding into the cytoplasm, since these residues contribute to, but do not account for, membrane binding. Surprisingly, modified forms of VP7 which are secreted from transfected cells showed the same membrane-binding properties in vitro as the protein retained in the ER membrane. Thus, secreted VP7 may not be present as a soluble polypeptide in the ER. A model to explain these results is presented. Previously published data are consistent with the idea that the highly conserved C terminus of nascent VP7 could have a cytoplasmic orientation which is important for assembly of mature virus particles.
Mutational analysis of the 18-base-pair inverted repeat element at the bovine papillomavirus origin of replication: identification of critical sequences for E1 binding and in vivo replication
Authors: Holt S E , Wilson V G
Published: January 2020

Replication of bovine papillomavirus requires two viral proteins, E1 and E2-TA. Previously we demonstrated that sequences within an imperfect 18-bp inverted repeat (IR) element were sufficient to confer specific binding of the E1 protein to the origin region (S. E. Holt, G. Schuller, and V. G. Wilson, J. Virol. 68:1094-1102, 1994). To identify critical nucleotides for E1 binding and origin function, a series of individual point mutations was constructed at each nucleotide position in the 18-bp IR. Binding of E1 to these point mutations established that both the position of the mutation and the specific nucleotide change were important for the E1-DNA interaction. Equivalent mutations from each half of the IR exhibited similar binding, suggesting that the halves were functionally symmetric for E1 interactions. Each of these mutations was evaluated also for origin function in vivo by a transient-replication assay. No single point mutation eliminated replication capacity completely, though many mutants were severely impaired, demonstrating an important functional contribution for the E1 binding site. Furthermore, E1 binding was not sufficient for replication, as several origin mutants bound E1 well in vitro but replicated poorly in vivo. This suggests that certain nucleotides within the 18-bp IR may be involved in postbinding events necessary for replication initiation. The results with the point mutations suggest that E1-E1 interactions are important for stable complex formation and also indicate that there is some flexibility with regard to formation of a functional E1 replication complex at the origin.
Genetic analysis of human immunodeficiency virus type 1 integrase and the U3 att site: unusual phenotype of mutants in the zinc finger-like domain
Authors: Masuda T , Planelles V , Krogstad P , Chen I S
Published: January 2020

Retroviral integration is the step which leads to establishment of the provirus, cis- and trans-acting regions of the human immunodeficiency type 1 (HIV-1) retrovirus genome, including the attachment site (att) at the ends of the unintegrated viral DNA and the conserved domains within the integrase (IN) protein, have been identified as being important for integration. We investigated the role of each of these regions in the context of an infectious HIV-1 molecular clone through point mutagenesis of the att site and the zinc finger-like and catalytic domains of IN. The effect of each mutation on integration activity was examined by using a single-step infection system with envelope-pseudotype virus. The relative integration efficiency was estimated by monitoring the levels of viral DNA over time in the infected cells. The integration activities of catalytic domain point mutants and att site deletion mutants were estimated to be 0.5 and 5% of wild-type activity, respectively. However, in contrast with previous in vitro cell-free integration studies, alteration of the highly conserved CA dinucleotide resulted in a mutant which still retained 40% of wild-type integration activity. The relative levels of expression of each mutant, as measured by a luciferase reporter gene, correlated with levels of integration. This observation is consistent with those of previous studies indicating that integration is an obligatory step for retroviral gene expression. Interestingly, we found that three different HIV-1 constructs bearing point mutations in the zinc finger-like domain synthesized much lower levels of viral DNA after infection, suggesting impairment of these mutants before or at the initiation of reverse transcription. Western blot (immunoblot) analysis demonstrated wild-type levels of reverse transcriptase within the mutant virions. In vitro endogenous reverse transcription assays indicated that all three mutants in the zinc finger-like domain had wild-type levels of reverse transcriptase activity. These data indicate that in addition to integration, IN may have an effect on the proper course of events in the viral life cycle that precede integration.
Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb, and p53, leading to cell cycle arrest
Authors: Jault F M , Jault J M , Ruchti F , Fortunato E A , Clark C , Corbeil J , Richman D D , Spector D H
Published: January 2020

Human cytomegalovirus (HCMV) infection stimulates cellular DNA synthesis and causes chromosomal damage. Because such events likely affect cellular proliferation, we investigated the impact of HCMV infection on key components of the cell cycle. Early after infection, HCMV induced elevated levels of cyclin E, cyclin E-associated kinase activity, and two tumor suppressor proteins, p53 and the retinoblastoma gene product (Rb). The steady-state concentration of Rb continued to rise throughout the infection, with most of the protein remaining in the highly phosphorylated form. At early times, HCMV infection also induced cyclin B accumulation, which was associated with a significant increase in mitosis-promoting factor activity as the infection progresses. In contrast, the levels of cyclin A and cyclin A-associated kinase activity increased only at late times in the infection, and the kinetics were delayed relative to those for cyclins E and B. Analysis of the cellular DNA content in the infected cells by flow cytometry showed a progressive shift of the cells from the G1 to the S and G2/M phases of the cell cycle, leading to an accumulation of aneuploid cells at late times. We propose that these HCMV-mediated perturbations result in cell cycle arrest in G2/M.
Role of the virion host shutoff (vhs) of herpes simplex virus type 1 in latency and pathogenesis
Authors: Strelow L I , Leib D A
Published: January 2020

The herpes simplex virus type 1 (HSV-1) UL41 gene product, virion host shutoff (vhs), has homologs among five alphaherpesviruses (HSV-1, HSV-2, pseudorabies virus, varicella-zoster virus, and equine herpesvirus 1), suggesting a role for this protein in neurotropism. A mutant virus, termed UL41NHB, which carries a nonsense linker in the UL41 open reading frame at amino acid position 238 was generated. UL41NHB and a marker-rescued virus, UL41NHB-R, were characterized in vitro and tested for their ability to replicate in vitro and in vivo and to establish and reactivate from latency in a mouse eye model. As demonstrated by Western blotting (immunoblotting) and Northern (RNA) blotting procedures, UL41NHB encodes an appropriately truncated vhs protein and, as expected for a vhs null mutant, fails to induce the degradation of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The growth of UL41NHB was not significantly altered in one-step growth curves in Vero or mouse C3H/10T1/2 cells but was impaired in corneas, in trigeminal ganglia, and in brains of mice compared with the growth of KOS and UL41NHB-R. As a measure of establishment of latency, quantitative DNA PCR showed that the amount of viral DNA within trigeminal ganglia latently infected with UL41NHB was reduced by approximately 30-fold compared with that in KOS-infected ganglia and by 50-fold compared with that in UL41NHB-R-infected ganglia. Explant cocultivation studies revealed a low reactivation frequency for UL41NHB (1 of 28 ganglia, or 4%) compared with that for KOS (56 of 76, or 74%) or UL41NHB-R (13 of 20 or 65%). Taken together, these results demonstrate that vhs represents a determinant of viral pathogenesis.
p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease
Authors: Huang M , Orenstein J M , Martin M A , Freed E O
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55Gag, contains at its C-terminal end a proline-rich, 6-kDa domain designated p6. Two functions have been proposed for p6: incorporation of the HIV-1 accessory protein Vpr into virus particles and virus particle production. To characterize the role of p6 in the HIV-1 life cycle and to map functional domains within p6, we introduced a number of nonsense and single and multiple amino acid substitution mutations into p6. Following the introduction of the mutations into the full-length HIV-1 molecular clone pNL4-3, the effects on Gag protein expression and processing, virus particle production, and virus infectivity were analyzed. The production of mutant virus particles was also examined by transmission electron microscopy. The results indicate that (i) p6 is required for efficient virus particle production from a full-length HIV-1 molecular clone; (ii) a Pro-Thr-Ala-Pro sequence, located between residues 7 and 10 of p6, is critical for virus particle production; (iii) mutations outside the Pro-Thr-Ala-Pro motif have little or no effect on virus assembly and release; (iv) the p6 defect is manifested at a late stage in the budding process; and (v) mutations in p6 that severely reduce virion production in HeLa cells also block or significantly delay the establishment of a productive infection in the CEM (12D-7) T-cell line. We further demonstrate that mutational inactivation of the viral protease reverses the p6 defect, suggesting a functional linkage between p6 and the proteolytic processing of the Gag precursor protein during the budding of progeny virions.
Characterization of double-stranded RNA satellites associated with the Trichomonas vaginalis virus
Authors: Khoshnan A , Alderete J F
Published: January 2020

Three small and distinct satellite double-stranded RNAs (dsRNAs) denoted s1, s1', and s2 were recently described for a Trichomonas vaginalis isolate harboring a dsRNA virus. Since characterization of these satellite dsRNAs might provide insight into the virus replication cycle and virus-host interactions, full-length cDNAs to s1 and s1' dsRNAs were synthesized and sequenced. s1 dsRNA has 688 bp, and s1' dsRNA has 616 bp. A 228-bp open reading frame that begins at nucleotide 37 was detected on a putative sense strand of s1. All satellite RNAs were found associated with RNA-dependent RNA polymerase (RDRP) activity that banded on CsCl gradients. Within carrier trichomonads, satellite RNAs synthesized single-stranded replicative intermediates. An in vitro assay was established to assess replication of satellite RNAs. Transcripts generated from s1 cDNA, for example, served as a template for the viral RDRP. These templates had a polarity similar to that of the replicative intermediate found in the satellite-harboring parasites. Importantly, the recognition of s1 RNA was shown to be specific, since unrelated RNAs did not serve as templates for RDRP under the same experimental conditions. The data indicate that the cDNA of s1 has a specific and essential sequence needed for recognition by the viral RDRP and for subsequent RNA synthesis. Both s1 and s1' have conserved domains, albeit of unproven function, but which may be required for replication.
Isolation and characterization of a new simian T-cell leukemia virus type 1 from naturally infected celebes macaques (Macaca tonkeana): complete nucleotide sequence and phylogenetic relationship with the Australo-Melanesian human T-cell leukemia virus type 1
Authors: Ibrahim F , de Thé G , Gessain A
Published: January 2020

A study of simian T-cell leukemia virus type 1 (STLV-1) infection in a captive colony of 23 Macaca tonkeana macaques indicated that 17 animals had high human T-cell leukemia virus type 1 (HTLV-1) antibody titers. Genealogical analysis suggested mainly a mother-to-offspring transmission of this STLV-1. Three long-term T-cell lines, established from peripheral blood mononuclear cell cultures from three STLV-1-seropositive monkeys, produced HTLV-1 Gag and Env antigens and retroviral particles. The first complete nucleotide sequence of an STLV-1 (9,025 bp), obtained for one of these isolates, indicated an overall genetic organization similar to that of HTLV-1 but with a nucleotide variability for the structural genes ranging from 7.8 to 13.1% compared with the HTLV-1 ATK and STLV-1 PTM3 Asian prototypes. The Tax and Rex regulatory proteins were well conserved, while the pX region, known to encode new proteins in HTLV-1 (open reading frames I and II), was more divergent than that in the ATK strain. Furthermore, a fragment of 522 bp of the gp21 env gene from uncultured peripheral blood mononuclear cell DNAs from five of the STLV-1-infected monkeys was sequenced. Phylogenetic trees constructed with the long terminal repeat and env (gp46 and gp21) regions demonstrated that this new STLV-1 occupies a unique position within the Asian STLV-1 and HTLV-1 isolates, being, by most analyses, related more to the Australo-Melanesian HTLV-1 topotype than to any other Asian STLV-1. These data raise new hypotheses on the possible interspecies viral transmission between monkeys carrying STLV-1 and early Australoid settlers, ancestors of the present day Australo-Melanesian inhabitants, during their migrations from the Southeast Asian land mass to the greater Australian continent.
Retrovirus infection: effect of time and target cell number
Authors: Morgan J R , LeDoux J M , Snow R G , Tompkins R G , Yarmush M L
Published: January 2020

Using a model amphotropic recombinant retrovirus encoding the Escherichia coli lacZ gene and quantitative assays to measure virus infection, we have determined the effects of time and target cell number on infectivity. Infection of various numbers of NIH 3T3 fibroblasts showed that the extent of lacZ virus infection was dependent on virus concentration and independent of target cell number. These results demonstrate that multiplicity of infection is not an accurate predictor of the efficiency of retroviral infection. Varying the time of viral infection revealed that maximal infection occurred after greater than 24 h of exposure of the cells to the lacZ virus. Half-maximal infection occurred after 5 h of exposure. After 2 h of adsorption at 37 degrees C, the majority of infectious virus was not adsorbed to cells but was unbound and able to infect other cells. These results are discussed in terms of both their relevance to the fundamental biology of retrovirus infection and the use of recombinant retroviruses for retrovirus-mediated gene transfer with purposes of gene therapy.
Persistent infection of macaques with simian-human immunodeficiency viruses
Authors: Li J T , Halloran M , Lord C I , Watson A , Ranchalis J , Fung M , Letvin N L , Sodroski J G
Published: January 2020

Chimeric simian-human immunodeficiency viruses (SHIV) containing the human immunodeficiency virus type 1 (HIV-1) tat, rev, env, and, in some cases, vpu genes were inoculated into eight cynomolgus monkeys. Viruses could be consistently recovered from the CD8-depleted peripheral blood lymphocytes of all eight animals for at least 2 months. After this time, virus isolation varied among the animals, with viruses continuing to be isolated from some animals beyond 600 days after inoculation. The level of viral RNA in plasma during acute infection and the frequency of virus isolation after the initial 2-month period were higher for the Vpu-positive viruses. All of the animals remained clinically healthy, and the absolute numbers of CD4-positive lymphocytes were stable. Antibodies capable of neutralizing HIV-1 were generated at high titers in animals exhibiting the greatest consistency of virus isolation. Strain-specific HIV-1-neutralizing antibodies were initially elicited, and then more broadly neutralizing antibodies were elicited. env sequences from two viruses isolated more than a year after infection were analyzed. In the Vpu-negative SHIV, for which virus loads were lower, a small amount of env variation, which did not correspond to that found in natural HIV-1 variants, was observed. By contrast, in the Vpu-positive virus, which was consistently isolated from the host animal, extensive variation of the envelope glycoproteins in the defined variable gp120 regions was observed. Escape from neutralization by CD4 binding site monoclonal antibodies was observed for the viruses with the latter envelope glycoproteins, and the mechanism of escape appears to involve decreased binding of the antibody to the monomeric gp120 glycoproteins. The consistency with which SHIV infection of cynomolgus monkeys is initiated and the similarities in the neutralizing antibody response to SHIV and HIV-1 support the utility of this model system for the study of HIV-1 prophylaxis.
Emergence of virus escape mutants after immunization with epitope vaccine
Authors: Weidt G , Deppert W , Utermöhlen O , Heukeshoven J , Lehmann-Grube F
Published: January 2020

BALB/c and C57BL/6J mice were immunized with recombinant vaccines consisting of lymphocytic choriomeningitis virus CD8+ T-lymphocyte epitopes and a carrier protein. During challenge infection with WE strain lymphocytic choriomeningitis virus, mutants with alterations in distinct amino acid residues of the epitopic nonapeptides appeared and multiplied. Splenocytes from WE-infected BALB/c mice lysed cells coated with the WE-type epitope; lysis was considerably less effective when the epitopic nonapeptide with which the syngeneic cells had been sensitized was the mutated form. Neither target was lysed by splenocytes from BALB/c mice infected with the variant virus. Mutants were not detected in F1 hybrid mice immunized with two viral epitopes that were restricted by class I molecules of both parents.
Hepatitis delta virus mutant: effect on RNA editing
Authors: Wu T T , Bichko V V , Ryu W S , Lemon S M , Taylor J M
Published: January 2020

During the replication cycle of hepatitis delta virus (HDV), RNA editing occurs at position 1012 on the 1679-nucleotide RNA genome. This changes an A to G in the amber termination codon, UAG, of the small form of the delta antigen (delta Ag). The resultant UGG codon, tryptophan, allows the translation of a larger form of the delta Ag with a 19-amino-acid C-terminal extension. Using HDV cDNA-transfected cells, we examined the editing potential of HDV RNA mutated from G to A at 1011 on the antigenome, adjacent to normal editing site at 1012. Four procedures were used to study not only the editing of the A at 1012, but also that of the new A at 1011: (i) nucleotide sequencing, (ii) a PCR-based RNA-editing assay, (iii) immunoblot assays, and (iv) immunofluorescence. Five findings are reported. (i) Even after the mutation at 1011, editing still occurred at 1012. (ii) Site 1011 itself now acted as a novel RNA-editing site. (iii) Sites 1011 and 1012 were edited independently. (iv) At later times, both sites became edited, thereby allowing the synthesis of the large form of the delta Ag (delta Ag-L). (v) Via immunofluorescence, such double editing became apparent as a stochastic event, in that groups of cells arose in which the changes had taken place. Evaluation of these findings and of those from previous studies of the stability of the HDV genomic sequence (H.J. Netter et al., J. Virol. 69:1687-1692, 1995) supports both the recent reevaluation of HDV RNA editing as occurring on antigenomic RNA (Casey and Gerin, personal communication) and the interpretation that editing occurs via the RNA-modifying enzyme known as DRADA.
Evidence that flavivirus NS1-NS2A cleavage is mediated by a membrane-bound host protease in the endoplasmic reticulum
Authors: Falgout B , Markoff L
Published: January 2020

Previous deletion mutagenesis studies have shown that the flavivirus NS1-NS2A clevage requires the eight C-terminal residues of NS1, constituting the cleavage recognition sequence, and sequences in NS2A far downstream of the cleavage site. We now demonstrate that replacement of all of NS1 upstream of the cleavage recognition sequence with prM sequences still allows cleavage in vivo. Thus, other than the eight C-terminal residues, NS1 is dispensable for NS1-NS2A cleavage. However, deletion of the N-terminal signal sequence abrogated cleavage, suggesting that entry into the exocytic pathway is required. Cleavage in vivo was not blocked by brefeldin A, and cleavage could occur in vitro in the presence of dog pancreas microsomes, indicating that NS1-NS2A cleavage occurs in the endoplasmic reticulum. Four in-frame deletions in NS2A were cleavage defective in vitro, as were two mutants in which NS4A-NS4B sequences were substituted for NS2A, suggesting that most of NS2A is required. A series of substitution mutants were constructed in which all Asp, Cys, Glu, His, and Ser residues in NS2A were collectively replaced; all standard proteases require at least one of these residues in their active sites. No single mutant was cleavage defective, suggesting that NS2A is not a protease. Fractionation of the microsomes indicated that the lumenal contents were not required for NS1-NS2A cleavage. It seems most likely that NS1-NS2A cleavage is effected by a host membrane-bound endoplasmic reticulum-resident protease, quite possibly signalase, and that NS2A is required to present the cleavage recognition sequence in the correct conformation to the host enzyme for cleavage.
Avian homologs of the mammalian low-density lipoprotein receptor family bind minor receptor group human rhinovirus
Authors: Gruenberger M , Wandl R , Nimpf J , Hiesberger T , Schneider W J , Kuechler E , Blaas D
Published: January 2020

Avian oocyte-specific very low density lipoprotein receptor specifically binds human rhinovirus of the minor receptor group on ligand blots and in solution. The solubilized receptor protects cell against infection in a dose-dependent manner.
Active foamy virus proteinase is essential for virus infectivity but not for formation of a Pol polyprotein
Authors: Konvalinka J , Löchelt M , Zentgraf H , Flügel R M , Kräusslich H G
Published: January 2020

To analyze proteolytic processing of foamy (spuma) retroviruses, two mutations were generated in the presumed active-site triplet Asp-Ser-Gly in the predicted proteinase (PR) region of the human foamy virus (HSRV). The mutations changed either the presumed catalytic aspartic acid residue to a catalytically incompetent alanine or the adjacent serine to a threonine found in most cellular and retroviral proteases at this position. Both mutations were cloned into the full-length infectious HSRV DNA clone. Wild-type and S/T mutant genomes directed the synthesis of particles with similar infectious titers, while the HSRV D/A PR mutant was noninfectious. Immunoblot analysis of transfected cells revealed identical patterns for the wild-type and for the S/T PR mutant. HSRV D/A mutant-transfected cells expressed only a single Gag polyprotein of 78 kDa instead of the 78-kDa-74-kDa doublet found in HSRV-infected or wild-type-transfected cells. Analysis with pol-specific antisera yielded a protein of approximately 120 kDa reactive with antisera against pol- but not gag-specific domains. No Gag-Pol polyprotein was detected in this study. Electron microscopy analysis of transfected cells showed heterogeneous particle morphology in the case of the D/A mutant, with particles of normal appearance and particles of aberrant size and shape. These results indicate that foamy viruses have an aspartic PR that is essential for infectivity but not for formation of the 120-kDa Pol polyprotein.
A novel V beta 2-specific endogenous mouse mammary tumor virus which is capable of producing a milk-borne exogenous virus
Authors: Niimi N , Wajjwalku W , Ando Y , Nakamura N , Ueda M , Yoshikai Y
Published: January 2020

We have previously reported new Mtv loci, Mtv-48 and -51, in the Japanese laboratory mouse strains CS and NC. Here we show by backcross analysis that both Mtv-48 and -51 cosegregate with very slow deletion of T cells bearing V beta 2. The nucleotide sequences of the open reading frames in the 3' long terminal repeats of Mtv-48 and -51 were very similar to those of Mtv-DDO, mouse mammary tumor virus C4 [MMTV(C4)], and MMTV(BALB/cV), which encode V beta 2-specific superantigens. Furthermore, backcross female mice carrying Mtv-48 but not Mtv-51 were found to be able to produce milk-borne MMTV(CS), which can vigorously stimulate V beta 2-expressing T cells after local injection in vivo in an I-E-dependent manner. On the other hand, mice carrying Mtv-51 but not Mtv-48 could not produce such an MMTV in milk. The nucleotide sequences of MMTV(CS) open reading frame were completely matched with those of Mtv-48. These results indicate that the provirus Mtv-48 but not Mtv-51 is capable of producing a milk-borne virus of which the superantigen stimulates V beta 2-expressing T cells.
Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence
Authors: Casal J I , Langeveld J P , Cortés E , Schaaper W W , van Dijk E , Vela C , Kamstrup S , Meloen R H
Published: January 2020

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.
Activation of human immunodeficiency virus type 1 in monocytoid cells by the protozoan parasite Leishmania donovani
Authors: Bernier R , Turco S J , Olivier M , Tremblay M
Published: January 2020

In this study, we demonstrated that the protozoan parasite Leishmania donovani and one of its major surface molecules, the lipophosphoglycan (LPG), can induce human immunodeficiency virus type 1 (HIV-1) expression in U1 and OM-10.1, two cell lines of monocytoid origin latently infected with HIV-1. Treatment of U1 cells with various concentrations of LPG (1, 5, and 10 microM) resulted in a dose-dependent secretion of tumor necrosis factor alpha (TNF-alpha). Suppression of LPG-induced HIV-1 expression by polyclonal anti-TNF-alpha antibodies further confirmed the involvement of this cytokine. Results from these studies indicate that the protozoan parasite L. donovani can induce the secretion of TNF-alpha that will function in an autocrine or paracrine manner to upregulate HIV-1 expression. Our data suggest for the first time that this protozoan parasite can be viewed as a potential cofactor in the pathogenesis of AIDS.
Initiator elements and regulated expression of the herpes simplex virus thymidine kinase gene
Authors: Cook W J , Lin S M , DeLuca N A , Coen D M
Published: January 2020

Regulated expression of thymidine kinase mRNA in herpes simplex virus mutants harboring thymidine kinase promoters that lacked functional TATA boxes was largely unaffected by additional sequence alterations around the transcriptional start site. A strong initiator element increased the regulated expression of a TATA-containing promoter by 50% but did not affect that of the TATA-less promoter. Thus, initiator elements exert only small effects in this promoter context.
Mode of spread to and within the central nervous system after oral infection of neonatal mice with the DA strain of Theiler's murine encephalomyelitis virus
Authors: Ha-Lee Y M , Dillon K , Kosaras B , Sidman R , Revell P , Fujinami R , Chow M
Published: January 2020

Theiler's murine encephalomyelitis virus is a neurotropic enterovirus known to cause biphasic neural disease after intracerebral inoculation into adult mice. The present study characterizes a neonatal mouse model with a high disease incidence for the study of the acute phase of the pathogenesis of the DA strain of Theiler's murine encephalomyelitis virus after oral infection. The route of viral spread to and within the central nervous system (CNS) was determined by examining the kinetics of viral replication in various organs and by performing histopathological analysis. Viral antigen was detected widely in the neonatal CNS, mainly in the gray matter, and it was asymmetrical and multifocal in its distribution, with considerable variation in lesion distribution from animal to animal. Necrotizing lesions appeared to expand by direct extension from infected cells to their close neighbors, with a general disregard of neuroanatomical boundaries. The diencephalon showed particular susceptibility to viral infection. Other areas of the CNS, including the cerebellum and dentate gyrus of the hippocampus, were consistently spared. Neurons with axons extending peripherally to other organs or receiving direct input from the peripheral nervous system were not preferentially affected. The kinetics of viral replication in the liver, spleen, and CNS and the histopathological findings indicate that viral entry to the CNS is via a direct hematogenous route in orally infected neonatal mice and that the disease then progresses within the CNS mainly by direct extension from initial foci.
Genesis of Sindbis virus by in vivo recombination of nonreplicative RNA precursors
Authors: Raju R , Subramaniam S V , Hajjou M
Published: January 2020

Genetically engineered RNA transcripts coding for various Sindbis virus (SIN) genes were used to study structure and sequence requirements of RNA recombination in BHK cells. Three different groups of RNA transcripts were made: (i) RNAs which retain the ability to replicate and which carry sequences coding for either viral polymerase or viral structural proteins; (ii) RNAs which lack the complete 3' end of the SIN genome and thus are incapable of replicating; and (iii) RNAs which lack the complete 5' end of the SIN genome and also are incapable of replicating. BHK cells were transfected with specific combinations of these precursor RNAs, and virus production and RNA synthetic abilities of the released virus were determined. We demonstrate in vivo generation of infectious SIN by fusion of (i) replicative RNAs to nonreplicative RNAs and (ii) two nonreplicative RNA precursors. Both homologous and nonhomologous types of recombinations were observed. In the homologous type of recombination, a 694-nucleotide overlap at the crossover region of the first pair of precursors resulted in the addition of an A residue converting the UAG stop codon of nonstructural protein P4 to a UAA stop codon. In the nonhomologous type of recombination, the crossover sites contained deletion of up to 76 nucleotides from one of the precursors and complete preservation of junction sequence from the other precursor. This is also the first report that a cytoplasmic RNA virus can be generated from biologically nonreplicative RNA precursors. These results have implications for initiation of viral RNA synthesis and recombination between RNA viral genomes in general. We favor template switching as a mechanism for the fusion events described here and suggest inclusion of polymerase scanning of diverse nonreplicative RNAs as an inherent feature of the copy choice model of RNA recombination. Very importantly, the facile nature of RNA recombination occurring between nonreplicative RNA precursors should speed up the production and analysis of targeted mutants of SIN and possibly other RNA viruses.
Adenovirus E1A243 disrupts the ATF/CREB-YY1 complex at the mouse c-fos promoter
Authors: Zhou Q , Engel D A
Published: January 2020

The adenovirus E1A243 protein can activate transcription of the mouse c-fos gene in a manner that depends on treatment of cells with inducers or analogs of cyclic AMP (cAMP). Activation requires conserved region 1 and the N-terminal domain of E1A243 and is mediated by a 22-bp E1A response element containing a cAMP response element (CRE) at -67 and a binding site for transcription factor YY1 at -54. In the absence of E1A243, YY1 represses CRE-dependent transcription of c-fos by physically interacting with ATF/CREB proteins bound to the -67 CRE. Here we present evidence that expression of E1A243 leads to relief of YY1-mediated repression by a disruption of the ATF/CREB-YY1 complex. Addition of E1A243 to in vitro binding assays prevented binding of ATF-2 to glutathione S-transferase-YY1. Similarly, expression of E1A243 in HeLa cells prevented the association of a YY1-VP16 fusion protein with endogenous ATF/CREB proteins bound to the -67 CRE of a transfected c-fosCAT reporter plasmid. In each case, the N-terminal domain of E1A243, which mediates a direct interaction with YY1, was responsible for disruption of the ATF/CREB-YY1 complex. On the basis of these and previously published results, we present a model for the synergistic transcriptional activation of the c-fos gene by E1A243 and cAMP.
A Tat-induced auto-up-regulatory loop for superactivation of the human immunodeficiency virus type 1 promoter
Authors: Biswas D K , Salas T R , Wang F , Ahlers C M , Dezube B J , Pardee A B
Published: January 2020

The virus-encoded Tat protein strongly activates transcription of human immunodeficiency virus (HIV). A well-recognized mechanism involves interaction of Tat with the nascent RNA transcript of the viral tar gene; mutation of tar greatly decreases activation by Tat. However, Tat still provides a low level of activation, demonstrating that it also has a tar-independent mode of action. We propose that this tar-independent mode of Tat action is through activation of gene transcription to produce tumor necrosis factor alpha. This cytokine and other compounds that activate NF-kappa B up-regulate the HIV promoter at a low level, similarly to the second Tat action. Through this mechanism, they also activate promoters of tumor necrosis factor alpha and other cytokines and thereby establish an auto-up-regulatory loop. Activated NF-kappa B motifs in the HIV promoter synergize with Tat/tar. Mutations of these motifs decrease activation by Tat to a few percent of the wild-type value. In cooperation, the two modes of activation by Tat (tar dependent and cytokine based) set up positive up-regulatory loops which greatly superactivate transcription of HIV. Agents that block these synergistic pathways at three different steps and are more inhibitory in combination than is any one alone have been found. Thereby, multidrug modalities for transcription of HIV are proposed for virus suppression.
Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation
Authors: Bartenschlager R , Lohmann V , Wilkinson T , Koch J O
Published: January 2020

Processing of the hepatitis C virus polyprotein is mediated by host cell signalases and at least two virally encoded proteinases. Of these, the serine-type proteinase encompassing the amino-terminal one-third of NS3 is responsible for cleavage at the four sites carboxy terminal of NS3. The activity of this proteinase is modulated by NS4A, a 54-amino-acid polyprotein cleavage product essential for processing at the NS3/4A, NS4A/4B, and NS4B/5A sites and enhancing cleavage efficiency between NS5A and NS5B. Using the vaccinia virus-T7 hybrid system to express hepatitis C virus polypeptides in BHK-21 cells, we studied the role of NS4A in proteinase activation. We found that the NS3 proteinase and NS4A form a stable complex when expressed as a single polyprotein or as separate molecules. Results from deletion mapping show that the minimal NS4A domain required for proteinase activation is located in the center of NS4A between amino acids 1675 and 1686 of the polyprotein. Amino acid substitutions within this domain destabilizing the NS3-NS4A complex also impair trans cleavage at the NS4A-dependent sites. Similarly, deletion of amino-terminal NS3 sequences impairs complex formation as well as cleavage at the NS4B/5A site but not at the NS4A-independent NS5A/5B site. These results suggest that a stable NS3-NS4A interaction is important for cleavage at the NS4A-dependent sites and that amino-terminal NS3 sequences and the central NS4A domain are directly involved in complex formation.
Convergent evolution within the V3 loop domain of human immunodeficiency virus type 1 in association with disease progression
Authors: Strunnikova N , Ray S C , Livingston R A , Rubalcaba E , Viscidi R P
Published: January 2020

Phylogenetic analysis was used to study in vivo genetic variation of the V3 region of human immunodeficiency virus type 1 in relation to disease progression in six infants with vertically acquired human immunodeficiency virus type 1 infection. Nucleotide sequences from each infant formed a monophyletic group with similar average branch lengths separating the sets of sequences. In contrast to the star-shaped phylogeny characteristic of interinfant viral evolution, the shape of the phylogeny formed by sequences from the infants who developed AIDS tended to be linear. A computer program, DISTRATE, was written to analyze changes in DNA distance values over time. For the six infants, the rate of divergence from the initial variant was inversely correlated with CD4 cell counts averaged over the first 11 to 15 months of life (r = -0.87, P = 0.024). To uncover evolutionary relationships that might be dictated by protein structure and function, tree-building methods were applied to inferred amino acid sequences. Trees constructed from the full-length protein fragment (92 amino acids) showed that viruses from each infant formed a monophyletic group. Unexpectedly, V3 loop protein sequences (35 amino acids) that were found at later time points from the two infants who developed AIDS clustered together. Furthermore, these sequences uniquely shared amino acids that have been shown to confer a T-cell line tropic phenotype. The evolutionary pattern suggests that viruses from these infants with AIDS acquired similar and possibly more virulent phenotypes.
The Vif protein of human and simian immunodeficiency viruses is packaged into virions and associates with viral core structures
Authors: Liu H , Wu X , Newman M , Shaw G M , Hahn B H , Kappes J C
Published: January 2020

The vif gene of human and simian immunodeficiency viruses (HIV and SIV) encodes a late gene product that is essential for viral infectivity in natural target cells. Virions produced in the absence of Vif are abnormal in their ultrastructural morphology and are severely impaired in the ability to complete proviral DNA synthesis upon entry into new target cells. Because previous studies failed to detect Vif protein in virus particles, Vif is believed to influence virus infectivity indirectly, by affecting virion assembly, release, and/or maturation. In this report, we reexamined the possibility that Vif is a virion-associated protein. Utilizing high-titer Vif-specific antibodies, a sensitive immunoblot technique, and highly concentrated virus preparations, we detected a 23-kDa Vif-reactive protein in wild-type HIV type 1 (HIV-1) and a 27-kDa Vif-reactive protein in wild-type SIVSM virions. Neither protein was present in virions derived from vif-deficient HIV-1 and SIVSM proviral constructs. Vif protein content was similar among different strains of HIV-1 and was independent of the cell type (permissive or nonpermissive) used to produce the virus. To determine the subvirion localization of Vif, HIV-1 virions were treated with proteinase K or Triton X-100 to remove virion surface proteins and the viral membrane, respectively, purified through sucrose, and analyzed by immunoblot analysis. Vif protein content was not affected by the removal of external surface proteins or by the removal of the viral membrane and submembrane p17Gag matrix protein. Instead, Vif colocalized with viral core structures which sedimented at a density of 1.25 g/ml on linear sucrose gradients (enveloped HIV-1 particles sediment at a density of 1.17 g/ml). Finally, the amount of Vif protein packaged into virions was estimated to be on the order of 1 molecule of Vif for every 20 to 30 molecules of p24Gag, or between 60 and 100 molecules of Vif per particle. These results indicate that Vif represents an integral component of HIV and SIV particles and raise the possibility that it plays a direct role in early replication events.