bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyomavirus DNA replication in the pancreas and in a transformed pancreas cell line has distinct enhancer requirements
Authors: Rochford R , Villarreal L P
Published: January 2020

The regulatory DNA (enhancer) of polyomavirus (Py) is a major determinant of tissue-specific DNA replication during acute infection of newborn mice. Previously, we reported that the combination of one of the two Py enhancers (A enhancer) and the repeated Moloney murine leukemia virus (Mo-MuLV) enhancer gave a chimeric Py genome (Py-MuLV) that replicates predominantly in the acinar cells of the pancreas, a tissue not permissive for wild-type PyA2 replication (R. Rochford, B. A. Campbell, and L. P. Villareal. Proc. Nat. Acad. Sci. USA 84:449-453,1987). In this report, we further examine the combined enhancer requirements for acinar cell-specific Py replication. We also compare enhancer requirements for Py replication in the acinar cells of the pancreas with those of a transformed acinar cell line (266-6 cells). The deletion of sequences within the A enhancer of Py-MuLV (nucleotides 5098 to 5132) results in a virus with 10-fold-reduced levels of pancreas-specific replication. The deletion, however, of one of the 72-bp repeated Mo-MuLV enhancer sequences from Py-MuLV results in a complete loss of pancreas-specific DNA replication. Thus, the Py A enhancer is required for efficient replication of Py in the pancreas without otherwise altering organ specificity, but both of the repeated copies of the Mo-MuLV enhancer are essential for pancreas-specific Py replication. In contrast to the enhancer requirements for in vivo pancreas replication, in transformed acinar cells (266-6), PyA2 wild-type replicated efficiently and the Py-MuLV recombinant replicated inefficiently. These data suggest that the cell-specific control of DNA replication is different between normal pancreas cells and their transformed cell line counterparts and that this difference is apparent in the enhancer requirement of cell-specific Py DNA replication.
Cooperative binding of the E2 protein of bovine papillomavirus to adjacent E2-responsive sequences
Authors: Monini P , Grossman S R , Pepinsky B , Androphy E J , Laimins L A
Published: January 2020

The DNA-binding properties of purified full-length E2 protein from bovine papillomavirus type 1 have been investigated by utilizing a quantitative gel shift analysis. By using a recombinant baculovirus which express the E2 open reading frame from the polyhedrin promoter, the full-length E2 protein was synthesized in insect cells and purified to homogeneity by using an E2 binding site (ACCGN4CGGT)-specific oligonucleotide column. The Kd of E2 binding to a 41-bp oligonucleotide containing a single binding site was found to be 2 x 10(-11) M. When two binding sites were included on an oligonucleotide, cooperative binding to these sites by the E2 protein was observed. A cooperativity parameter of 8.5 was determined for E2 binding to two sites. An 86-amino-acid peptide encompassing the C terminus of the protein retains the ability to bind E2 binding sites with a Kd of 4 x 10(-10) M but exhibits slight cooperativity of binding to two adjacent sites. A major determinant for cooperative binding of the full-length E2 protein is thus encoded by the N-terminal amino acids outside the minimal DNA binding domain.
Mutational analysis of the carboxyl terminus of the Moloney murine leukemia virus integration protein
Authors: Roth M J
Published: January 2020

The integration protein (IN) is required for retrovirus DNA integration into the host DNA. The function of the C terminus of the Moloney murine leukemia virus IN protein was examined. The terminal 28 amino acids were found to be nonessential. A linker insertion at position 6025, within a 36-amino-acid insertion not found in any other retrovirus, also produced viable virus.
Evidence that less-than-full-length pol gene products are functional in hepadnavirus DNA synthesis
Authors: Wu T T , Condreay L D , Coates L , Aldrich C , Mason W
Published: January 2020

Duck hepatitis B virus mutants containing frameshift or stop codon mutations in a portion of the viral pol gene separating the terminal protein and reverse transcriptase domains had a leaky phenotype and, depending on the location and type of mutation, synthesized up to 10% as much viral DNA as did the wild type. This region of the pol gene had previously been reported to be refractory to missense mutations; in fact, the leakiness of most of our mutants appeared attributable to translational suppression, which would also be expected to introduce amino acid changes. However, at least one mutant (pH1093 + 2), which was ca. 10% as active as the wild type, appeared to use a novel pathway to express the viral pol gene. Our analyses indicated that pH1093 + 2 synthesized the viral reverse transcriptase as a fusion protein with the amino-terminal portion of the pre-S envelope protein. Thus, in this case, the products of the terminal-protein and reverse transcriptase domains of the pol gene would function as separate protein species, though perhaps noncovalently joined in a dimeric structure during assembly of DNA replication complexes. Evidence was also obtained that was consistent with the idea that the wild-type pol gene may, at least in certain instances, be expressed as functional, subgenic polypeptides.
Assembly and transcription of synthetic vesicular stomatitis virus nucleocapsids
Authors: Moyer S A , Smallwood-Kentro S , Haddad A , Prevec L
Published: January 2020

The functional template for transcription of vesicular stomatitis virus (VSV) RNA is a ribonucleoprotein particle (nucleocapsid) consisting of the negative-strand sense genomic RNA completely encapsidated by the viral nucleocapsid (N) protein. As an approach to create nucleocapsids in vitro, we demonstrate here the specific encapsidation by purified N protein of in vitro-synthesized RNA sequences representing the 5' end of both the negative- and positive-strand VSV genome-length RNAs. As few as 19 nucleotides from the 5'-end of positive-strand RNA allowed maximal encapsidation, although the 5' terminal 10 nucleotides would allow partial (50%) encapsidation. Sequences downstream of the binding site can be of any origin. Specific encapsidation of VSV sequences was dependent on the presence of uninfected cell cytoplasmic extracts or poly(A). The synthetic nucleocapsids have the properties of RNase resistance and a buoyant density typical of wild-type VSV nucleocapsids. We have encapsidated a synthetic virionlike RNA species which contained just the terminal sequences of the virion RNA: the N encapsidation signal from the 5' end and the leader gene from the 3' end. This assembled nucleocapsid could function in vitro as a transcription template for the VSV RNA polymerase.
Human immunodeficiency virus type 1 infection of human placenta: potential route for fetal infection
Authors: Amirhessami-Aghili N , Spector S A
Published: January 2020

To determine the potential role of the placenta in transmission of human immunodeficiency virus (HIV) from mother to fetus, the ability of human placental tissue to support HIV type 1 (HIV-1) infection was examined. HIV-1-seronegative first-trimester placentas were maintained in culture and infected with HIV-1. Virus production, measured by HIV-1 antigen release into the supernatant, and HIV-1 DNA, identified by polymerase chain reaction, were detected for at least 12 days postinfection. Western immunoblot analysis showed Gag proteins, precursor p55, and cleavage products p24 and p17 in HIV-1-infected tissues. Double labeling of placental villi with antibodies to CD4 and placental trophoblast-specific alkaline phosphatase indicated that trophoblasts express CD4 antigen. Additionally, immunostaining of HIV-1-infected tissues with anti-p24 antibodies demonstrated HIV-1 protein expression in placental trophoblasts. Evaluation of human chorionic gonadotropin and progesterone production by the placental cultures indicated that there was a 90% decrease in human chorionic gonadotropin and a 70% decrease in progesterone production in HIV-1-infected cultures in comparison with controls. These data demonstrate that trophoblastic cells of human placenta tissue express CD4 and are susceptible to HIV-1 infection; also, placental endocrine function is decreased by HIV-1 infection. Thus, the placenta may serve as a reservoir of HIV-1 infection during pregnancy contributing to infection of the fetus, and decreased placental hormone production may result in impaired fetal development.
Effects of chimeric mutants of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex on nucleolar targeting signals
Authors: Kubota S , Nosaka T , Cullen B R , Maki M , Hatanaka M
Published: January 2020

Two chimeric mutant genes derived from rev of human immunodeficiency virus type 1 and rex of human T-cell leukemia virus type I were constructed to investigate the functions of the nucleolar-targeting signals (NOS) in Rev and Rex proteins. A chimeric Rex protein whose NOS region was substituted with the NOS of Rev was located predominantly in the cell nucleolus and functioned like the wild-type protein in the Rex assay system. However, a chimeric Rev with the NOS of Rex abolished Rev function despite its nucleolar localization. This nonfunctional nucleolar-targeting chimeric protein inhibited the function of both Rex and Rev. In the same experimental conditions, this mutant interfered with the localization of the functional Rex in the nucleolus.
Regulation of the herpesvirus saimiri (HVS) delayed-early 110-kilodalton promoter by HVS immediate-early gene products and a homolog of the Epstein-Barr virus R trans activator
Authors: Nicholas J , Coles L S , Newman C , Honess R W
Published: January 2020

We have reported previously the detection of two stable immediate-early (IE) transcripts that accumulate in cycloheximide-treated cells infected with herpesvirus saimiri (HVS). These are the 1.6-kb mRNA from the 52-kDa gene (which is homologous to the BSLF2-BMLF1 gene of Epstein-Barr virus) and the 1.3-kb mRNA from the HindIII-G fragment of virus DNA. In order to study the roles of the HVS IE gene products in the progression of a lytic infection, the promoter region of the delayed-early 110-kDa gene of HVS was sequenced, the transcription initiation site was mapped by RNase protection, and the promoter sequences were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene. Sequences between -447 and +37 (relative to the 110-kDa transcription initiation site) were sufficient for response to HVS superinfection of transfected cells, but the 110-kDa promoter was activated only poorly by the 52-kDa and HindIII-G IE (IE-G) proteins in cotransfection experiments. However, a distinct region of the genome, EcoRI-D (15 kbp), was able to activate 110-kDa-CAT expression relatively efficiently in similar experiments. A 4.7-kbp PstI fragment encoding this function was isolated and sequenced, and further subcloning identified the gene encoding the EcoRI-D trans activator. This gene, which we now designate HVS.R, is homologous to the BRLF1-encoded transcriptional effector of Epstein-Barr virus.
Serologic and mucosal immune response to rotavirus infection in the rabbit model
Authors: Conner M E , Gilger M A , Estes M K , Graham D Y
Published: January 2020

We examined the humoral immune response to rotavirus infection in specific pathogen-free rabbits inoculated and challenged orally with rabbit Ala rotavirus (7.5 x 10(5) to 1 x 10(7) PFU). The humoral immune response in both serologic and mucosal samples was monitored by using total antibody enzyme-linked immunosorbent assays (ELISAs), isotype-specific ELISAs, and plaque reduction neutralization assays. Following a primary infection, all rabbits shed virus and serologic and mucosal antibody responses were initially detected by 1 week postinoculation. Intestinal immunoglobulin M was detected by 3 days postinoculation, and secretory immunoglobulin A was detected by 6 days postinoculation. Following challenge, rabbits were protected (no detectable virus shedding) from infection. An anamnestic immune response was observed only with mucosal neutralizing antibodies, and all serologic and mucosal immune responses persisted at high levels until at least 175 days postchallenge (204 days postinoculation). Detection of neutralization responses was influenced by the virus strain used in the neutralization assay; all inoculated rabbits developed detectable serum and intestinal neutralizing antibodies against the infecting (Ala) virus strain. Neutralization activity in both serum and mucosal samples was generally, but not exclusively, homotypic (VP7 serotype 3) after both primary and challenge inoculations with Ala virus. Heterotypic serum neutralization activity was observed with serotype 8 (9 of 12 rabbits) and 9 (12 of 12 rabbits) viruses and may be based on reactivity with the outer capsid protein VP4 or on a shared epitope in the C region of VP7. Comparisons of heterologous (serotype 3) and heterotypic neutralizing responses in mucosal and serologic samples revealed that 43% (21 of 49) of the responses were discordant. In 19 of 49 (39%) of these cases, a heterotypic serologic response was seen in the absence of a heterotypic mucosal response, but in 2 of 49 (4%) instances, a heterotypic mucosal response was seen in the absence of a concomitant serologic response. These results provide insight into factors which may affect detection of heterotypic responses.
An antigen encoded by the latency-associated transcript in neuronal cell cultures latently infected with herpes simplex virus type 1
Authors: Doerig C , Pizer L I , Wilcox C L
Published: January 2020

During latent infection of neurons with herpes simplex virus type 1, viral transcription is restricted to the latency-associated transcripts (LATs). These RNAs contain open reading frames, but detection of a protein encoded by the LATs has not been reported. We used immunocytochemical techniques to demonstrate that an antiserum directed against a bacterially expressed fusion protein containing part of a LAT-encoded polypeptide recognized an antigen present in primary neurons latently infected in vitro. This antigen (called LAA, for latency-associated antigen) was not detected in mock-infected neurons, in productively infected Vero cells, or in neurons latently infected with a mutant virus carrying a deletion in the LAT gene. By Western immunoblot analysis, we demonstrated the presence of a protein with an apparent molecular mass of 80 kDa recognized by the anti-LAA antiserum in latently infected neurons.
Differentiation of human embryonal carcinoma cells induces human immunodeficiency virus permissiveness which is stimulated by human cytomegalovirus coinfection
Authors: Hirka G , Prakash K , Kawashima H , Plotkin S A , Andrews P W , Gönczöl E
Published: January 2020

Human immunodeficiency virus (HIV) replicates in differentiated but not undifferentiated NTERA-2 human embryonal carcinoma cells; neither cell type expresses CD4. Susceptibility of the differentiated cells is enhanced by coinfection with cytomegalovirus. HIV infection induces lactoseries glycolipids, suggesting a mechanism whereby HIV might interfere with normal embryogenesis.
Expression of herpes simplex virus type 2 latency-associated transcript in neurons and nonneurons
Authors: Tenser R B , Edris W A , Hay K A , de Galan B E
Published: January 2020

The presence of herpes simplex virus type 2 (HSV-2) transcription during in vivo latent infection was investigated by in situ hybridization. Latent infection of mouse dorsal root ganglion was investigated with the BamHI p fragment of HSV-2, which resulted in evidence of ganglion hybridization, and other fragments representing approximately 40% of the genome, which did not result in hybridization. Strand specificity of hybridization was investigated in studies with synthetic oligonucleotides, which supported the conclusion that a latency-associated transcript(s) had been detected. Hybridization was detected with oligonucleotides complementary to the infected-cell polypeptide 0 (ICP0) template strand but not with oligonucleotides synthesized from the ICP0 template strand. Although most hybridization occurred over neurons, in some instances hybridization appeared to occur over nonneuronal ganglion cells, and this was more evident when tissue sections were examined by phase contrast microscopy. Although these results supported the usual neuronal site of HSV-2 latency, latency in nonneuronal cells may be important in considering the pathobiology of HSV-2 infections.
A 269-amino-acid segment with a pseudo-leucine zipper and a helix-turn-helix motif codes for the sequence-specific DNA-binding domain of herpes simplex virus type 1 origin-binding protein
Authors: Deb S , Deb S P
Published: January 2020

The UL9 gene of herpes simplex virus (HSV) codes for a DNA-binding protein (OBP) that interacts sequence specifically with the origin of replication. This protein is essential for HSV DNA replication in cultured cells. The UL9 gene was cloned into a plasmid vector downstream of the SP6 RNA polymerase promoter. By using in vitro transcription and translation systems, a full-length OBP was synthesized. This synthetic protein is recognized by an antiserum generated against the C-terminal decapeptide of OBP and is functionally active in binding to OriS sequence specifically. The in vitro-synthesized protein has sequence specificity for binding similar to that found for the in vivo-generated OBP. A total of 14 in-frame deletion and insertion mutants of the UL9 gene were generated and expressed in vitro. Using these deletion mutants, we determined that the 269-amino-acid stretch defined by amino acids 564 to 832 localizes the OriS-specific DNA-binding domain. The N-terminal boundary is between amino acids 565 and 596, while the C terminus lies between amino acids 833 and 805. This segment contains a helix-turn-helix moiety and a pseudo-leucine zipper, neither of which alone can support DNA binding. The other leucine zipper from amino acids 150 to 173 is not required for the in vitro sequence-specific DNA-binding activity of OBP.
Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase
Authors: Sankar S , Porter A G
Published: January 2020

Encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which allowed easy purification of the fusion protein by affinity chromatography on immobilized glutathione. Inclusion of a thrombin cleavage site between the GST carrier and the viral enzyme facilitated the release of purified mature EMC virus RNA polymerase from the GST carrier by proteolysis with thrombin. The purified recombinant enzyme has a molecular mass of about 52 kDa and is recognized by polyclonal immune serum raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme comigrates with immunoprecipitated EMC virus RNA polymerase from infected mouse L929 cell extracts when run in parallel lanes on a sodium dodecyl sulfate-polyacrylamide gel. The enzyme exhibits rifampin-resistant, poly(A)-dependent poly(U) polymerase activity and RNA polymerase activity, which are both oligo(U) dependent. Template-size products are synthesized in in vitro reactions with EMC virus genomic RNA or globin mRNA. The availability of recombinant EMC virus RNA polymerase in a purified form will allow biochemical analysis of its role in the replication of the virus as well as structure-function studies of this unique class of enzyme.
Expression of active human immunodeficiency virus type 1 protease by noninfectious chimeric virus particles
Authors: Kohl N E , Diehl R E , Rands E , Davis L J , Hanobik M G , Wolanski B , Dixon R A
Published: January 2020

To generate nonpathogenic viral particles which express active human immunodeficiency virus type 1 (HIV-1) protease (PR), plasmids containing sequences from the genomes of HIV-1 and Moloney murine leukemia virus (M-MuLV) were constructed. Either the PR coding region alone; the gag, PR, and reverse transcriptase protein-coding regions; or the complete gag and pol protein-coding regions from HIV-1 were substituted for the corresponding regions of a full-length M-MuLV clone to yield the chimeric plasmids pMoHIV-I, pMoHIV-III, and pMoHIV-IV, respectively. Cell lines which express the viral gag polyprotein were isolated for hybrids pMoHIV-I and pMoHIV-III. These cells produced viral particles which contained processed core proteins. Cleavage of the gag polyprotein in the viral particles was inhibited by the HIV-1 PR inhibitor L-687908, indicating that the viral PR is responsible for the observed processing. The hybrid virions were not infectious; analyses indicated that the viral particles contained little or no reverse transcriptase activity. In addition, particles produced by pMoHIV-III transfectants failed to package the viral genomic RNA. The cell line which expresses and processes the HIV-1 gag polyprotein is a safe and effective reagent for the in vivo evaluation of potential inhibitors of the HIV-1 PR.
Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle
Authors: Noteborn M H , de Boer G F , van Roozelaar D J , Karreman C , Kranenburg O , Vos J G , Jeurissen S H , Hoeben R C , Zantema A , Koch G
Published: January 2020

Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained double- and single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.
Immediate-early gene expression is sufficient for induction of natural killer cell-mediated lysis of herpes simplex virus type 1-infected fibroblasts
Authors: Fitzgerald-Bocarsly P , Howell D M , Pettera L , Tehrani S , Lopez C
Published: January 2020

Herpes simplex virus type 1 (HSV-1)-infected human fibroblast (HSV-FS) targets are susceptible to lysis by natural killer (NK) cells, whereas uninfected FS are resistant to lysis. Studies were undertaken to determine the mechanism of this preferential susceptibility. HSV-FS were not intrinsically less stable than FS, as determined by a 51Cr release assay under hypotonic shock in the presence of rat granule cytolysin and by sensitivity to anti-human leukocyte antigen class I antibody plus complement. Single-cell assays in agarose demonstrated that although similar numbers of large granular lymphocytes bound to the HSV-FS and FS targets, the conjugates with HSV-FS were lysed at a much higher frequency than those with FS. These results suggested that both targets are bound by the NK cells but only the HSV-FS were able to trigger lysis. The requirement for active virus expression was demonstrated by failure of emetine-treated HSV-FS targets or targets infected with UV-inactivated HSV to be lysed by NK effectors. To evaluate the role of viral glycoproteins in conferring susceptibility to lysis, Fab were prepared from HSV-1-seropositive sera; these Fab were unable to block lysis of the HSV-FS. Furthermore, incubation in phosphonoacetic acid failed to reduce NK(HSV-FS) activity despite sharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissive temperature were lysed as well as or better than targets infected with wild-type virus, indicating that HSV immediate-early gene product expression is sufficient for conferring susceptibility to lysis. We conclude that expression of nonstructural viral proteins or virally induced cellular gene products early in the course of infection rather than structural glycoproteins is required for NK lysis of HSV-FS targets.
Improvement of avian leukosis virus (ALV)-based retrovirus vectors by using different cis-acting sequences from ALVs
Authors: Cosset F L , Legras C , Thomas J L , Molina R M , Chebloune Y , Faure C , Nigon V M , Verdier G
Published: January 2020

Production and expression of double-expression vectors which transduce both Neo(r) and lacZ genes and are based on the structure of avian leukosis virus were enhanced by using cis-acting sequences (long terminal repeats and noncoding sequences) from Rous-associated virus-1 and Rous-associated virus-2 rather than those of avian erythroblastosis virus previously used in our constructs. Polyclonal producer cells obtained after transfection of these vectors into the Isolde packaging cell line gave rise to titers as high as 3 x 10(5) lacZ CFU/ml, whereas it was possible to isolate clones of producer cells giving rise to titers of more than 10(6) resistance focus-forming units per ml.
Characterization of V protein in measles virus-infected cells
Authors: Wardrop E A , Briedis D J
Published: January 2020

An edited mRNA transcribed from the phosphoprotein (P) gene of measles virus (MV) has been predicted to encode a cysteine-rich protein designated V. This mRNA contains a single additional nontemplated G residue which permits access to an additional protein-coding reading frame. Such an edited P gene-specific mRNA has been detected in MV-infected cells, but no corresponding protein has yet been identified in vivo. We report the use of antisera directed against synthetic peptides corresponding to five different regions of the predicted MV V protein amino acid sequence to analyse MV-specific proteins synthesized in vivo and in vitro. The MV V protein (40 kDa) was detected in MV-infected cells in a diffuse cytoplasmic distribution, a predominant subcellular localization distinct from that of virus nucleocapsids. The protein was found to be phosphorylated and to be maximally synthesized at 16 h postinfection, when MV-specific structural protein synthesis was also maximal. Antiserum directed against a peptide (PV2) corresponding to amino acids 65 to 87 of the V protein amino acid recognized the P protein but not the V protein, indicating that the P and V proteins may be folded differently at or near this region so that the PV2 sequence is in an exposed position at the surface of the P protein but not at the surface of the V protein.
Simian immunodeficiency virus negative factor suppresses the level of viral mRNA in COS cells
Authors: Niederman T M , Hu W , Ratner L
Published: January 2020

The nef gene is conserved among all human and simian lentiviruses. However, the amino acid similarity between simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 NEF is only 38%. To assess the role of SIV NEF on virus replication and compare its activity with that of its human immunodeficiency virus type 1 counterpart, we examined the activity of an intact nef gene from proviral clone pSIV 102, an isolate from SIV-MAC-251-infected cells. Proviral clone pSIV BA was constructed by introducing a premature termination codon at codon 40 of the nef gene without altering the predicted amino acid sequence of the overlapping env gene. These two clones were transfected into CD4- COS cells, and virus replication was monitored by p27 enzyme-linked immunosorbent assay kits. In seven independent experiments, clone pSIV BA afforded two- to sixfold greater levels of viral antigen compared with those in clone pSIV 102 and two- to sixfold-increased levels of viral mRNAs as indicated with Northern (RNA) blot and S1 nuclease protection analyses. Nuclear run-on assays demonstrated a two- to threefold increased rate of RNA synthesis with nuclei isolated from cells transfected with pSIV BA compared with that from cells transfected with pSIV 102. In contrast, there was no apparent destabilization of SIV mRNAs by NEF, as measured in dactinomycin-treated cells. This study demonstrates that SIV NEF is a negative regulator of virus replication and acts by suppressing the level of mRNA synthesis and accumulation in COS cells.
A transcription factor for expression of vaccinia virus late genes is encoded by an intermediate gene
Authors: Wright C F , Keck J G , Tsai M M , Moss B
Published: January 2020

A factor, designated VLTF-1, that is required in vitro for specific transcription of vaccinia virus late genes was previously isolated from vaccinia virus-infected cells. Subsequent genetic experiments identified three vaccinia virus genes, encoding proteins of 17, 26, and 30 kDa, that together trans activate late gene expression in vivo. The purpose of this study was to determine whether VLTF-1 corresponded to one of the three trans activators. Toward this end, VLTF-1 was further purified, the trans-activator genes were expressed in Escherichia coli, and antisera were made to the native and recombinant proteins. Antibody to the 30-kDa recombinant protein reacted on Western immunoblots with a protein of approximately Mr 30,000 that cochromatographed and cosedimented with VLTF-1 activity from virus-infected cells. Conversely, antibody to purified VLTF-1 bound to products produced by in vitro transcription and translation of the open reading frame encoding the 30-kDa trans-activator protein. Both antisera depleted VLTF-1 activity and blocked late gene transcription by partially purified extracts of vaccinia virus-infected cells. Taken together, these data demonstrate that the 30-kDa trans activator comprises part, if not all, of VLTF-1 activity.
Human immunodeficiency virus type 1 NL4-3 replication in four T-cell lines: rate and efficiency of entry, a major determinant of permissiveness
Authors: Srivastava K K , Fernandez-Larsson R , Zinkus D M , Robinson H L
Published: January 2020

Single-cycle infections have been used to study the human immunodeficiency virus type 1 (HIV-1) life cycle in CD4+ T-cell lines that differ in their permissiveness for infection. In single-cycle infections of highly permissive C8166 cells, 50% of the infectious units escaped being blocked by a monoclonal antibody against the virus binding site on CD4 (leu3a) within 30 min. In contrast, 50% of the infectious units for three less permissive cell lines (H9, A3.01, and Jurkat) required 4 h to escape the leu3a block. Entry was also more efficient in the highly permissive cells, with NL4-3 stocks having three times more infectious units for C8166 cells than for H9, A3.01, or Jurkat cells. Postentry steps up through reverse transcription required approximately 3.5 h in each of the cell lines. The times lapsing between reverse transcription and the expression of reverse transcripts ranged from 17 to 25 h in the different cell lines. Virus production per cell was also similar in the different cell lines (within 1.5-fold of each other). These results indicate that a major determinant of the permissiveness of growing T cells for HIV-1 is the rate and efficiency of virus entry.
An HLA-C-restricted CD8+ cytotoxic T-lymphocyte clone recognizes a highly conserved epitope on human immunodeficiency virus type 1 gag
Authors: Littaua R A , Oldstone M B , Takeda A , Debouck C , Wong J T , Tuazon C U , Moss B , Kievits F , Ennis F A
Published: January 2020

A unique epitope on the gag protein of human immunodeficiency virus type 1 (HIV-1), located at amino acid 145 to 150, has been mapped by using a CD8+ cytotoxic T-lymphocyte (CTL) clone. This epitope is highly conserved among 18 HIV-1 strains. The HIV-1 gag-specific human leukocyte antigen (HLA) class I-restricted CD8+ CTL clone was generated from fresh peripheral blood mononuclear cells of an HIV-seropositive donor by stimulation with gamma-irradiated allogeneic peripheral blood mononuclear cells in the presence of an anti-CD3 monoclonal antibody and recombinant interleukin-2. This gag-specific CTL clone killed autologous target cells infected with a recombinant vaccinia virus containing the gag gene of HIV-1 and target cells pulsed with an authentic p24gag construct expressed in Escherichia coli. Fine specificity was determined by using a panel of overlapping 30-amino-acid-long synthetic peptides and subsequently using smaller peptides to precisely map the CTL domain on p24. The epitope is on a highly conserved region, and it overlaps with a major B-cell epitope of gag. This CD8+ T-cell epitope is restricted by HLA-Cw3, which has not been previously identified as a restricting element for human CTL responses.
The antigen of hepatitis delta virus: examination of in vitro RNA-binding specificity
Authors: Chao M , Hsieh S Y , Taylor J
Published: January 2020

The only known protein of hepatitis delta virus (HDV), the delta antigen, is found both within virus particles and within the nucleus of the infected cell, where it has one or more roles essential for RNA genome replication. Others have demonstrated that the antigen has the ability, in vitro, to specifically bind HDV RNA species. We report a further examination of this phenomenon, using partially purified recombinant protein, expressed as a fusion with the staphylococcal protein A. From Northwestern (RNA-immunoblot) analyses with both complete and various subdomains of HDV genomic and antigenomic RNAs, we found that a necessary feature for specific binding was that the RNA be able to fold to some extent into the so-called rodlike structure; this structure is a predicted intramolecular partial base-pairing of the circular RNA, with about 70% of all bases involved, so as to produce an unbranched rodlike structure. Six different subregions of the HDV rodlike structure, three on the genomic RNA and three on its complement, the antigenomic RNA, were tested and found to be sufficient for antigen binding. However, features in addition to the rodlike structure may also be necessary for specific binding, because we found that a similar structure present in the RNA of the potato spindle tuber viroid did not allow binding.
Generation and analysis of nonhomologous RNA-RNA recombinants in brome mosaic virus: sequence complementarities at crossover sites
Authors: Bujarski J J , Dzianott A M
Published: January 2020

All three single-stranded RNAs of the brome mosaic virus (BMV) genome contain a highly conserved, 193-base 3' noncoding region. To study the recombination between individual BMV RNA components, barley plants were infected with a mixture of in vitro-transcribed wild-type BMV RNAs 1 and 2 and an RNA3 mutant that carried a deletion near the 3' end. This generated a population of both homologous and nonhomologous 3' recombinant BMV RNA3 variants. Sequencing revealed that these recombinants were derived by either single or double crossovers with BMV RNA1 or RNA2. The primary sequences at recombinant junctions did not show any similarity. However, they could be aligned to form double-stranded heteroduplexes. This suggested that local hybridizations among BMV RNAs may support intermolecular exchanges.
Characterization of variable regions in the envelope and S3 open reading frame of equine infectious anemia virus
Authors: Alexandersen S , Carpenter S
Published: January 2020

The polymerase chain reaction was used to amplify and clone parts of the envelope gene and overlapping S3 open reading frame, thought to encode rev, of the virulent in vivo-derived Th-1 isolate of equine infectious anemia virus (EIAV). The results indicated that EIAV consists of a heterogeneous mixture of genotypes present at the first febrile cycle after initial infection. We showed that the Th-1 isolate apparently contains nondefective genotypes as well as types which have transmembrane protein truncations or are rev deficient. Furthermore, we could confirm the presence of a hypervariable region in the gp90 envelope glycoprotein. Taken together with earlier data on the heterogeneity of the regulatory motifs present in the long terminal repeat sequences of viruses from the same in vivo isolate (S. Carpenter, S. Alexandersen, M. J. Long, S. Perryman, and B. Chesebro, J. Virol. 65:1605-1610, 1991), our findings indicate that EIAV uses a complex system of diversity in biological phenotypes together with variation in regulatory and antigenic makeup to evade host response and to cause persistent infection and recurrent chronic disease.
Isolation of a simian immunodeficiency virus related to human immunodeficiency virus type 2 from a west African pet sooty mangabey
Authors: Marx P A , Li Y , Lerche N W , Sutjipto S , Gettie A , Yee J A , Brotman B H , Prince A M , Hanson A , Webster R G
Published: January 2020

Two of 25 healthy pet sooty mangabey (SM) monkeys (Cercocebus atys) living in West Africa were seropositive by immunoblot when surveyed for antibody to simian immunodeficiency virus of macaques (SIVmac). SIVsmLIB1 was isolated from one of the pet sooty mangabeys. Nucleotide sequence data showed that this isolate is a member of the SIVsm/human immunodeficiecy virus type 2 (HIV-2)/SIVmac group of primate lentiviruses. Furthermore, sequence comparisons revealed extensive genetic diversity among SIVsm isolates similar to that observed previously in SIV isolates from naturally infected African green monkeys. These observations provide additional evidence for monkey-human cross-species transmission of SIVsm as the source of HIV-2 infection of human.
Inhibition of vesicular stomatitis virus mRNA synthesis by human MxA protein
Authors: Staeheli P , Pavlovic J
Published: January 2020

The interferon-induced human MxA protein inhibits the multiplication of influenza virus and vesicular stomatitis virus (VSV) by an unknown mechanism. Here we show that MxA protein interferes with VSV mRNA synthesis. Transfected Swiss 3T3 mouse cells constitutively expressing MxA protein and control cells were infected with VSV, and viral RNA and protein synthesis was monitored. Viral macromolecules were very abundant in control cells at 4 h postinfection, whereas the pools of VSV proteins and RNAs were more than 50-fold reduced in cells expressing MxA. To determine whether MxA inhibited VSV primary transcription, we infected the cells in the presence of the protein synthesis inhibitor cycloheximide and measured the pools of the five viral mRNAs at 4 h postinfection. VSV L mRNA concentration was more than 20-fold reduced, VSV G mRNA concentration was about 10-fold reduced, and the other viral mRNAs were three- to fivefold less abundant in MxA-expressing cells than in control cells. Our results thus indicate that MxA interferes with normal VSV mRNA synthesis either directly by inhibiting the activity of the viral polymerase complex or indirectly by reducing the stability of the VSV mRNAs.
Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants
Authors: Strauss E G , Stec D S , Schmaljohn A L , Strauss J H
Published: January 2020

To study important epitopes on glycoprotein E2 of Sindbis virus, eight variants selected to be singly or multiply resistant to six neutralizing monoclonal antibodies reactive against E2, as well as four revertants which had regained sensitivity to neutralization, were sequenced throughout the E2 region. To study antigenic determinants in glycoprotein E1, four variants selected for resistance to a neutralizing monoclonal antibody reactive with E1 were sequenced throughout the E2 and E1 regions. All of the salient changes in E2 occurred within a relatively small region between amino acids 181 and 216, a domain that encompasses a glycosylation site at residue 196 and that is rich in charged amino acids. Almost all variants had a change in charge, suggesting that the charged nature of this domain is important for interaction with antibodies. Variants independently isolated for resistance to the same antibody were usually altered in the same amino acid, and reversion to sensitivity occurred at the sites of the original mutations, but did not always restore the parental amino acid. The characteristics of this region suggest that this domain is found on the surface of E2 and constitutes a prominent antigenic domain that interacts directly with neutralizing antibodies. Previous studies have shown that this domain is also important for penetration of cells and for virulence of the virus. Resistance to the single E1-specific neutralizing monoclonal antibody resulted from changes of Gly-132 of E1 to either Arg or Glu. Analogous to the findings with E2, these changes result in a change in charge and are found near a glycosylation site at residue 139. This domain of E1 may therefore be found near the 181 to 216 domain of E2 on the surface of the E1-E2 heterodimer; together, they could form a domain important in virus penetration and neutralization.
Identification of T- and B-cell epitopes of the E7 protein of human papillomavirus type 16
Authors: Comerford S A , McCance D J , Dougan G , Tite J P
Published: January 2020

There is strong evidence implicating human papillomavirus type 16 (HPV16) in the genesis of human genital cancer. Viral DNA has been identified in invasive carcinoma of the uterine cervix and in cell lines derived from cervical carcinomas. These sequences are actively transcribed, and translation products corresponding to the early (E)-region genes have been identified. The most abundant viral protein is the E7 protein, which has been shown to possess transforming activity for both established and primary cells. In addition, it has been shown to bind to a cellular tumor suppressor, the retinoblastoma gene product (pRb-105). In view of these properties, we have undertaken the immunological analysis of this protein and have identified four T-cell epitopes and three B-cell epitopes by using a series of overlapping peptides spanning the entire HPV16 E7 sequence. Two of the B-cell epitopes were recognized by antisera from mice with three different murine (H-2) haplotypes (k, d, and s) immunized with two different E7 fusion proteins and from Fischer rats seeded with baby rat kidney cells transformed by HPV16 E7 and ras. A third B-cell epitope was recognized by antisera from CBA mice seeded with HPV16 E7-expressing L cells. Two regions of the protein contain common B- and T-cell epitopes, one of which appears to be particularly immunodominant.
Flavivirus enzyme-substrate interactions studied with chimeric proteinases: identification of an intragenic locus important for substrate recognition
Authors: Preugschat F , Lenches E M , Strauss J H
Published: January 2020

The proteins of flaviviruses are translated as a single long polyprotein which is co- and posttranslationally processed by both cellular and viral proteinases. We have studied the processing of flavivirus polyproteins in vitro by a viral proteinase located within protein NS3 that cleaves at least three sites within the nonstructural region of the polyprotein, acting primarily autocatalytically. Recombinant polyproteins in which part of the polyprotein is derived from yellow fever virus and part from dengue virus were used. We found that polyproteins containing the yellow fever virus cleavage sites were processed efficiently by the yellow fever virus enzyme, by the dengue virus enzyme, and by various chimeric enzymes. In contrast, dengue virus cleavage sites were cleaved inefficiently by the dengue virus enzyme and not at all by the yellow fever virus enzyme. Studies with chimeric proteinases and with site-directed mutants provided evidence for a direct interaction between the cleavage sites and the proposed substrate-binding pocket of the enzyme. We also found that the efficiency and order of processing could be altered by site-directed mutagenesis of the proposed substrate-binding pocket.
Characterization of large deletions occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer
Authors: Pulsinelli G A , Temin H M
Published: January 2020

Retroviruses mutate at a high rate during replication. We used a spleen necrosis virus-based vector system and helper cell line to characterize mutations occurring during a single round of retrovirus replication. The vector used, JD216HyNeo, codes for two drug resistance genes, hygromycin resistance (hygro) and neomycin resistance (neo). The downstream neo gene is expressed only when a mutation alleviates a block to splicing which is located in the upstream hygro gene. The mutations allowing splicing were large deletions, ranging in size from about 500 to about 2,000 bp. Most of the mutant proviruses lacked the encapsidation sequence, as shown by our inability to rescue the mutant proviruses with wild-type reticuloendotheliosis virus strain A and confirmed by Southern blotting and direct DNA sequence analysis. We therefore concluded that most of the deletions arose during reverse transcription in the target cell, rather than during transcription in the host cell. The sequence data also indicated that the deletions occurred by at least three different mechanisms: (i) misalignment of the growing point; (ii) incorrect synthesis and termination in the primer-binding sequence during synthesis of the plus-strand strong-stop DNA; and (iii) incorrect synthesis and termination before the primer-binding sequence during synthesis of the plus-strand strong-stop DNA. The second mechanism also led to the incorporation of cellular sequences into the proviral genome, pointing to a potential novel mechanism by which retroviruses can acquire cellular genes.
Complex regulation of transcription from the hepatitis B virus major surface antigen promoter in human hepatoma cell lines
Authors: Raney A K , Milich D R , McLachlan A
Published: January 2020

A detailed mutational analysis of the regulatory DNA sequence elements that control expression of the hepatitis B virus major surface antigen gene was performed in the human hepatoma cell lines HepG2.1 and Huh7, using transient transfection assays. Seven regions (A to G) of the major surface antigen promoter located within 200 nucleotides of the RNA initiation site have been identified which influence the level of transcription from this promoter. The three distal regions (A to C), located between -188 and -68, appear to possess a level of redundancy in their ability to influence the transcriptional activity from the major surface antigen promoter. The simultaneous deletion of regions A, B, and C resulted in an approximately fourfold reduction in transcription from the major surface antigen promoter. Region D, located between -67 and -49, is an essential element of the major surface antigen promoter. The three proximal regions (E to G) are located within 45 nucleotides of the major transcription initiation site. Region E prevents the negative influence of region F and can compensate for the effect of mutation of region G on transcription from the major surface antigen promoter. Region G can compensate for the effect of the loss of a functional region E sequence on the transcriptional activity of the major surface antigen promoter only in the absence of a functional region F sequence. These results imply that the level of expression of the major surface antigen gene is controlled by the complex interplay between a minimum of six transcription factors which activate and one transcription factor which represses transcription from this gene.