bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteolytic processing of the replicase ORF1a protein of equine arteritis virus
Authors: Snijder E J , Wassenaar A L , Spaan W J
Published: January 2020

To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire ORF1a protein, results in cleavage products of approximately 29, 61, 22, 31, 41, and 3 kDa, which were named nonstructural proteins (nsps) 1 through 6, respectively. Pulse-chase experiments revealed that the cleavages at the nsp1/2 and nsp2/3 junctions are the most rapid processing steps. The remaining nsp3456 precursor is first cleaved at the nsp4/5 site. Final processing of the nsp34 and nsp56 intermediates is extremely slow. As predicted from previous in vitro translation experiments (E. J. Snijder, A. L. M. Wassenaar, and W. J. M. Spaan, J. Virol. 66:7040-7048, 1992), a cysteine protease domain in nsp1 was shown to be responsible for the nsp1/2 cleavage. The other processing steps are carried out by the putative EAV serine protease in nsp4 and by a third protease, which remains to be identified.
Processing of the intracellular form of the west Nile virus capsid protein by the viral NS2B-NS3 protease: an in vitro study
Authors: Yamshchikov V F , Compans R W
Published: January 2020

According to the existing model of flavivirus polyprotein processing, one of the cleavages in the amino-terminal part of the flavivirus polyprotein by host cell signalases results in formation of prM (precursor to one of the structural proteins, M) and the membrane-bound intracellular form of the viral capsid protein (Cint) retaining the prM signal sequence at its carboxy terminus. This hydrophobic anchor is subsequently removed by the viral protease, resulting in formation of the mature viral capsid protein found in virions (Cvir). We have prepared in vitro expression cassettes coding for both forms of the capsid protein, for the prM protein, for the C-prM precursor, and for the viral protease components of West Nile flavivirus and characterized their translation products. Using Cint and Cvir translation products as molecular markers, we have observed processing of the intracellular form of the West Nile capsid protein by the viral protease in vitro both upon cotranslation of the C-prM precursor and the viral protease-encoding cassette and by incubation of C-prM translation products with a detergent-solubilized extract of cells infected with a recombinant vaccinia virus expressing the active viral protease. The cleavage of Cint by the viral protease at the predicted dibasic site was verified by introduction of point mutations into the cleavage site and an adjacent region. These studies provide the first direct demonstration of processing of the intracellular form of the flavivirus capsid protein by the viral protease.
A 100-kilodalton polypeptide encoded by open reading frame (ORF) 1b of the coronavirus infectious bronchitis virus is processed by ORF 1a products
Authors: Liu D X , Brierley I , Tibbles K W , Brown T D
Published: January 2020

The genome-length mRNA (mRNA 1) of the coronavirus infectious bronchitis virus (IBV) contains two large open reading frames (ORFs), 1a and 1b, with the potential to encode polypeptides of 441 and 300 kDa, respectively. The downstream ORF, ORF 1b, is expressed by a ribosomal frameshifting mechanism. In an effort to detect viral polypeptides encoded by ORF 1b in virus-infected cells, immunoprecipitations were carried out with a panel of region-specific antisera. A polypeptide of approximately 100 kDa was precipitated from IBV-infected, but not mock-infected, Vero cells by one of these antisera (V58). Antiserum V58 was raised against a bacterially expressed fusion protein containing polypeptide sequences encoded by ORF 1b nucleotides 14492 to 15520; it recognizes specifically the corresponding in vitro-synthesized target protein. A polypeptide comigrating with the 100,000-molecular-weight protein (100K protein) identified in infected cells was also detected when the IBV sequence from nucleotides 8693 to 16980 was expressed in Vero cells by using a vaccinia virus-T7 expression system. Deletion analysis revealed that the sequence encoding the C terminus of the 100K polypeptide lies close to nucleotide 15120; it may therefore be generated by proteolysis at a potential QS cleavage site encoded by nucleotides 15129 to 15135. In contrast, expression of IBV sequences from nucleotides 10752 to 16980 generated two polypeptides of approximately 62 and 235 kDa, which represent the ORF 1a stop product and the 1a-1b fused product generated by a frameshifting mechanism, respectively, but no processed products were observed. Since the putative picornavirus 3C-like proteinase domain is located in ORF 1a between nucleotides 8937 and 9357, this observation suggests that deletion of the picornavirus 3C-like proteinase domain and surrounding regions abolishes processing of the 1b polyprotein. In addition, the in vitro translation and in vivo transfection studies also indicate that the ORF 1a region between nucleotides 8763 and 10720 contains elements that down-regulate the expression of ORF 1b.
Woodchuck hepatitis virus infections: very rapid recovery after a prolonged viremia and infection of virtually every hepatocyte
Authors: Kajino K , Jilbert A R , Saputelli J , Aldrich C E , Cullen J , Mason W S
Published: January 2020

Earlier studies have suggested that transient hepadnavirus infections in mammals are associated with virus replication in a large fraction of hepatocytes. Although the viremia that occurred during transient infections in some individuals would presumably lead to virus replication in all hepatocytes, these studies did not reveal if this was the case. The question of the extent of hepatocyte infection was therefore reinvestigated because of the implications of the results for the mechanisms of virus clearance. Woodchucks were inoculated with woodchuck hepatitis virus, and the course of hepatic infection was determined. These studies indicated that essentially 100% of the hepatocytes became infected in the majority of woodchucks. In 7 of 10 woodchucks, the viral infection was then rapidly cleared from the liver, generally in less than 4 weeks. In another three woodchucks, though productive infection was just as rapidly cleared, viral covalently closed circular DNA remained for weeks to months after other indicators of virus infection had disappeared from the liver. Bromodeoxyuridine labeling and anti-proliferating cell nuclear antigen staining to detect hepatocytes passing through S phase indicated an increase in hepatocyte proliferation during the recovery phase of infection. The rate of cell division appeared to be sufficient to replace no more than 2 to 3% of the hepatocytes per day, at the times at which the biopsies were performed. Histopathologic evaluation of the biopsy samples did not provide evidence for a massive amount of liver regeneration. Models to explain virus clearance, with or without massive immune system-mediated destruction of infected hepatocytes, are reviewed.
Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol
Authors: Neufeld K L , Galarza J M , Richards O C , Summers D F , Ehrenfeld E
Published: January 2020

A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA.
The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding
Authors: Engelman A , Hickman A B , Craigie R
Published: January 2020

The integrase protein of human immunodeficiency virus type 1 removes two nucleotides from the 3' ends of reverse-transcribed human immunodeficiency virus type 1 DNA (3' processing) and covalently inserts the processed ends into a target DNA (DNA strand transfer). Mutant integrase proteins that lack the amino-and/or carboxyl-terminal domains are incapable of catalyzing 3' processing and DNA strand transfer but are competent for an apparent reversal of the DNA strand transfer reaction (disintegration) in vitro. Here, we investigate the binding of integrase to DNA by UV cross-linking. Cross-linked complexes form with a variety of DNA substrates independent of the presence of divalent metal ion. Analysis with amino- and carboxyl-terminal deletion mutant proteins shows that residues 213 to 266 of the 288-residue protein are required for efficient cross-linking in the absence of divalent metal ion. Carboxyl-terminal deletion mutants that lack this region efficiently cross-link only to the branched disintegration DNA substrate, and this reaction is dependent on the presence of metal ion. Both the core and C-terminal domains of integrase therefore contribute to nonspecific DNA binding.
A lion lentivirus related to feline immunodeficiency virus: epidemiologic and phylogenetic aspects
Authors: Brown E W , Yuhki N , Packer C , O'Brien S J
Published: January 2020

Feline immunodeficiency virus (FIV) is a novel lentivirus that is genetically homologous and functionally analogous to the human AIDS viruses, human immunodeficiency virus types 1 and 2. FIV causes immunosuppression in domestic cats by destroying the CD4 T-lymphocyte subsets in infected hosts. A serological survey of over 400 free-ranging African and Asian lions (Panthera leo) for antibodies to FIV revealed endemic lentivirus prevalence with an incidence of seropositivity as high as 90%. A lion lentivirus (FIV-Ple) was isolated by infection of lion lymphocytes in vitro. Seroconversion was documented in two Serengeti lions, and discordance of mother-cub serological status argues against maternal transmission (in favor of horizontal spread) as a major route of infection among lions. A phylogenetic analysis of cloned FIV-Ple pol gene sequences from 27 lions from four African populations (from the Serengeti reserve, Ngorongoro Crater, Lake Manyara, and Kruger Park) revealed remarkably high intra- and interindividual genetic diversity at the sequence level. Three FIV-Ple phylogenetic clusters or clades were resolved with phenetic, parsimony, and likelihood analytical procedures. The three clades, which occurred not only together in the same population but throughout Africa, were as divergent from each other as were homologous pol sequences of lentivirus isolated from distinct feline species, i.e., puma and domestic cat. The FIV-Ple clades, however, were more closely related to each other than to other feline lentiviruses (monophyletic for lion species), suggesting that the ancestors of FIV-Ple evolved in allopatric (geographically isolated) lion populations that converged recently. To date, there is no clear evidence of FIV-Ple-associated pathology, raising the possibility of a historic genetic accommodation of the lion lentivirus and its host leading to a coevolved host-parasite symbiosis (or commensalism) in the population similar to that hypothesized for endemic simian immunodeficiency virus without pathology in free-ranging African monkey species.
Efficiency and selectivity of RNA packaging by Rous sarcoma virus Gag deletion mutants
Authors: Sakalian M , Wills J W , Vogt V M
Published: January 2020

In all retrovirus systems studied, the leader region of the RNA contains a cis-acting sequence called psi that is required for packaging the viral RNA genome. Since the pol and env genes are dispensable for formation of RNA-containing particles, the gag gene product must have an RNA binding domain(s) capable of recognizing psi. To gain information about which portion(s) of Gag is required for RNA packaging in the avian sarcoma and leukemia virus system, we utilized a series of gag deletion mutants that retain the ability to assemble virus-like particles. COS cells were cotransfected with these mutant DNAs plus a tester DNA containing psi, and incorporation of RNA into particles were measured by RNase protection. The efficiency of packaging was determined by normalization of the amount of psi+ RNA to the amount of Gag protein released in virus-like particles. Specificity of packaging was determined by comparisons of psi+ and psi- RNA in particles and in cells. The results indicate that much of the MA domain, much of the p10 domain, half of the CA domain, and the entire PR domain of Gag are unnecessary for efficient packaging. In addition, none of these deleted regions is needed for specific selection of the psi RNA. Deletions within the NC domain, as expected, reduce or eliminate both the efficiency and the specificity of packaging. Among mutants that retain the ability to package, a deletion within the CA domain (which includes the major homology region) is the least efficient. We also examined particles of the well-known packaging mutant SE21Q1b. The data suggest that the random RNA packaging behavior of this mutant is not due to a specific defect but rather is the result of the cumulative effect of many point mutations throughout the gag gene.
Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein
Authors: Ward P , Urcelay E , Kotin R , Safer B , Berns K I
Published: January 2020

The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication. An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication. In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay. Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA. When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated. When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue. There was no evidence that replication could initiate on the products of rescue. Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site. Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus. When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA. Replication was linear for 4 h, during which time 70% of the maximal synthesis took place. An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis.
The sequences of and distance between two cis-acting signals determine the efficiency of ribosomal frameshifting in human immunodeficiency virus type 1 and human T-cell leukemia virus type II in vivo
Authors: Kollmus H , Honigman A , Panet A , Hauser H
Published: January 2020

We have analyzed in cell culture the sequence elements that control the level of ribosomal frameshifting in the human T-cell leukemia virus type II (HTLV-2) gag-pro junction. The slippery sequence of HTLV-2 is sufficient to dictate a basal level of frameshifting. This level is enhanced by its upstream sequence context and by the downstream stem-loop structure which is located at an optimal distance of 7 bases. Frameshifting in human immunodeficiency virus gag-pol is similar to that of HTLV-2 gag-pro. However, experiments using hybrid cassettes of HTLV-2 and human immunodeficiency virus type 1 frameshift elements show that while the slippery sequence of HTLV-2 is less efficient, the stem-loop structure is a more efficient enhancer.
The adenovirus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane
Authors: Stewart A R , Tollefson A E , Krajcsi P , Yei S P , Wold W S
Published: January 2020

The adenovirus type 2 and 5 E3 10,400- and 14,500-molecular-weight (10.4K and 14.5K) proteins are both required to protect some cell lines from lysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor. We have shown previously that both 10.4K and 14.5K are integral membrane proteins and that 14.5K is phosphorylated and O glycosylated. The 10.4K protein coimmunoprecipitates with 14.5K, indicating that the two proteins function as a complex. Here we show, using immunofluorescence and two different cell surface-labeling techniques, that both proteins are localized in the plasma membrane. In addition, we show that trafficking of each protein to the plasma membrane depends on concomitant expression of the other protein. Finally, neither protein could be immunoprecipitated from conditioned media, indicating that neither is secreted. Taken together, these results suggest that the plasma membrane is the site at which 10.4K and 14.5K function to inhibit cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor.
Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV
Authors: Yoo B J , Selby M J , Choe J , Suh B S , Choi S H , Joh J S , Nuovo G J , Lee H S , Houghton M , Han J H
Published: January 2020

T7 RNA polymerase transcripts of a putative full-length cDNA clone of hepatitis C virus type 1 (HCV-1) were used to transfect a differentiated human hepatoma cell line, Huh7. The transfected genome replicated in cells, as evidenced by the appearance of progeny HCV RNA, detection of negative-strand viral RNA, and incorporation of [3H]uridine into the viral genome. Incubation of naive Huh7 cells with conditioned medium from transfected cells resulted in a new HCV infection, suggesting the production of biologically active virus in the inoculum. Maintenance of the transfected cells under serum-free culture conditions resulted in the selection of persistently infected cells which displayed a distinctive cellular morphology. This is the first demonstration that HCV RNA produced from cloned HCV cDNA is infectious and replication competent. This approach should provide a valuable system for studying HCV replication, persistence, and pathogenicity.
Human endogenous retrovirus K10: expression of Gag protein and detection of antibodies in patients with seminomas
Authors: Sauter M , Schommer S , Kremmer E , Remberger K , Dölken G , Lemm I , Buck M , Best B , Neumann-Haefelin D , Mueller-Lantzsch N
Published: January 2020

The human endogenous retrovirus K10 (HERV-K10) has been identified in the human genome by its homology to retroviruses of other vertebrates (M. Ono, T. Yasunaga, T. Miyata, and H. Ushikubo, J. Virol. 60:589-598, 1986). Using PCR amplification, DNA cloning, sequencing, and procaryotic expression, we were able to demonstrate that HERV-K10 encodes a 73-kDa protein which was processed by a HERV-K10-encoded protease to yield proteins p22/p26, p30, and p15/16. Analysis of the teratocarcinoma cell line Tera 1 or tumor tissues by immunoblotting demonstrated that the 80-kDa polyprotein of HERV-K10 gag and a processed protein of 39 kDa were expressed. In addition, a major protein of 39 kDa and additional species of 30, 22, 19, and 17 kDa could be detected in the supernatant of Tera 1 cells, suggesting that HERV-K10 Gag proteins are either secreted or processed to probably incomplete viral particles. In addition, the gag gene of HERV-K10 was expressed in the baculovirus system. Using this recombinant system to test antisera from patients with different diseases and healthy individuals, we were able to detect antibodies against the N-terminal part of HERV-K10 Gag in 2 to 4% of groups of tumor patients with titers ranging between 1:80 and 1:640, while approximately 0.1 to 0.5% of healthy individuals exhibited antibodies with lower titers. In contrast, patients with seminoma had antibody titers in the range of 1:2,560 at the time when the tumor was detected. Immunohistochemistry using specific rabbit sera or monoclonal antibodies against HERV-K10 Gag revealed that the Gag protein is expressed in the cytoplasm of the tumor cells. Furthermore, an 80-kDa protein corresponding to the HERV-K10 Gag polyprotein could be detected in tumor biopsies. For the first time, these data indicate that HERV-K10 Gag proteins are synthesized in seminoma cells and tumors exhibit relatively high antibody titers against Gag. So far, no information on which role HERV-K10 plays in the development of this tumor exists.
Disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity
Authors: Faaberg K S , Even C , Palmer G A , Plagemann P G
Published: January 2020

Disulfide bonds were found to link the nonglycosylated envelope protein VP-2/M (19 kDa), encoded by open reading frame 6, and the major envelope glycoprotein VP-3 (25 to 42 kDa), encoded by open reading frame 5, of lactate dehydrogenase-elevating virus (LDV). The two proteins comigrated in a complex of 45 to 55 kDa when the virion proteins were electrophoresed under nonreducing conditions but dissociated under reducing conditions. Furthermore, VP-2/M was quantitatively precipitated along with VP-3 in this complex by three neutralizing monoclonal antibodies to VP-3. The infectivity of LDV was rapidly and irreversibly lost during incubation with 5 to 10 mM dithiothreitol (> 99% in 6 h at room temperature), which is known to reduce disulfide bonds. LDV inactivation correlated with dissociation of VP-2/M and VP-3. The results suggest that disulfide bonds between VP-2/M and VP-3 are important for LDV infectivity. Hydrophobic moment analyses of the predicted proteins suggest that VP-2/M and VP-3 both possess three adjacent transmembrane segments and only very short ectodomains (10 and 32 amino acids, respectively) with one and two cysteines, respectively. Inactivation of LDV by dithiothreitol and dissociation of the two envelope proteins were not associated with alterations in LDV's density or sedimentation coefficient.
Inhibition of dengue virus by novel, modified antisense oligonucleotides
Authors: Raviprakash K , Liu K , Matteucci M , Wagner R , Riffenburgh R , Carl M
Published: January 2020

Five different target regions along the length of the dengue virus type 2 genome were compared for inhibition of the virus following intracellular injection of the cognate antisense oligonucleotides and their analogs. Unmodified phosphodiester oligonucleotides as well as the corresponding phosphorothioate oligonucleotides were ineffective in bringing about a significant inhibition of the virus. Novel modified phosphorothioate oligonucleotides in which the C-5 atoms of uridines and cytidines were replaced by propynyl groups caused a significant inhibition of the virus. Antisense oligonucleotide directed against the target region near the translation initiation site of dengue virus RNA was the most effective, followed by antisense oligonucleotide directed against a target in the 3' untranslated region of the virus RNA. It is suggested that the inhibitory effect of these novel modified oligonucleotides is due to their increased affinity for the target sequences and that they probably function via an RNase H cleavage of the oligonucleotide:RNA heteroduplex.
Characterization of nef sequences in long-term survivors of human immunodeficiency virus type 1 infection
Authors: Huang Y , Zhang L , Ho D D
Published: January 2020

Studies with the simian immunodeficiency virus have shown that nef deletion results in a low level of viremia and a lack of disease progression in monkeys. Given the similarity of this clinical profile to that observed in long-term survivors of human immunodeficiency virus type 1 (HIV-1) infection, we sought to examine the nef gene in 10 patients who are clinically healthy and immunologically normal despite 12 to 15 years of infection. PCR and DNA sequencing were used to determine nef sequences in peripheral blood mononuclear cells obtained from long-term survivors. We found that there is no gross deletion within nef in the cases studied; most nef sequences (91.1%) obtained from 10 subjects contained a full-length and intact open reading frame. In addition, at the protein level, there were no discernible differences between the Nef consensus sequences derived from long-term survivors and those from patients with AIDS. We therefore conclude that deletion of or gross sequence abnormality within nef is not likely to be a common explanation for the well-being of long-term survivors of HIV-1 infection. Moreover, phylogenetic analysis of nef sequences suggests that HIV-1 strains found in our study subjects do not have a common origin.
Nucleic acid vaccination primes hepatitis B virus surface antigen-specific cytotoxic T lymphocytes in nonresponder mice
Authors: Schirmbeck R , Böhm W , Ando K , Chisari F V , Reimann J
Published: January 2020

The efficiency of different vaccination techniques to prime in vivo major histocompatibility complex class I-restricted murine cytotoxic T-lymphocyte (CTL) precursors to hepatitis B virus small surface antigen (HBsAg) was investigated. Mice were immunized either by injection of a low dose of recombinant HBsAg protein preparations (native HBsAg particles or denatured HBsAg monomers) without adjuvants, by infection with recombinant vaccinia virus carrying an HBsAg-encoding gene, or by intramuscular transfer of plasmid DNA encoding HBsAg under appropriate promoter control. In H-2d mice, an Ld-restricted, S28-39-specific CTL response was efficiently primed by all alternative vaccination techniques tested, but the most potent priming of class I-restricted CTL to HBsAg in vivo was observed with DNA immunization. Priming of anti-HBsAg CTL in H-2b mice was not detectable after infection with a recombinant vaccinia virus or after injection with exogenous recombinant HBsAg preparations. After DNA immunization, however, both Kb- and Db-restricted CTL reactivity to HBsAg emerged in H-2b mice. Hence, nucleic acid immunization revealed class I-restricted CTL responsiveness to HBsAg in a mouse strain previously considered to be a nonresponder at the CTL level. These results demonstrate that the simple technique of nucleic acid immunization not only is extremely efficient but also reveals an extended spectrum of potentially immunogenic epitopes of protein antigens.
The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture
Authors: Schmolke S , Kern H F , Drescher P , Jahn G , Plachter B
Published: January 2020

The phosphoprotein pp65 (ppUL83) of human cytomegalovirus (HCMV) is abundantly synthesized during lytic infection in cultured fibroblasts. As a major constituent of extracellular particles, it gains entry to infected cells immediately after adsorption and subsequently translocates to the cell nucleus. This efficient transport is mediated by unique nuclear localization signals. To study the function of pp65, a viral deletion mutant was constructed by replacing the pp65 gene with the bacterial neomycin phosphotransferase gene, driven by the simian virus 40 early promoter. The resulting virus, RVAd65, could be grown and selected on human fibroblasts without complementation. The deletion of the pp65 gene in RVAd65 was verified by using Southern blot and PCR analyses. The lack of expression from the gene was investigated by immunoblotting with pp65-specific monoclonal antibodies. Single-cycle growth analyses showed that RVAd65 grew to levels of infectivity comparable to those of the wild-type virus. Therefore, pp65 is nonessential for the growth of HCMV in human fibroblasts. Electron microscopy revealed no differences in the processes of virion morphogenesis, although the maturation appeared to be delayed. However, the kinetics of expression of the immediate-early genes UL122 and UL123, the early gene UL44, and the late gene UL32 were the same in RVAd65-infected cells as in wild-type virus-infected cells in immunoblot analyses. In vitro phosphorylation assays showed that some of the virion proteins were labelled to a markedly reduced extent by virion-associated kinases in RVAd65 compared with wild-type virus. We therefore conclude that although deletion of the pp65 gene does not abolish replication of HCMV, a recombinant virus lacking pp65 displays phenotypic alterations compared with wild-type virus during growth in cultured fibroblasts.
Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine
Authors: Subbarao E K , Park E J , Lawson C M , Chen A Y , Murphy B R
Published: January 2020

We have previously described a strategy for the recovery of a synthetic influenza A virus wild-type (wt) PB2 gene (derived from influenza A/Ann Arbor/6/60 [AA] virus) into an infectious virus. It was possible to introduce an attenuating temperature-sensitive (ts) mutation at amino acid residue 265 of the AA wt PB2 gene and to rescue this mutant gene into infectious virus. Application of this new technology to influenza A virus vaccine development requires that multiple attenuating mutations be introduced to achieve a satisfactorily attenuated virus that retains the attenuation (att) phenotype following replication in vivo. In this report, we demonstrate that putative ts mutations at amino acids 112, 556, and 658 each indeed specify the ts and att phenotypes. Each of these mutations was introduced into a cDNA copy of the AA mutant mt265 PB2 gene to produce three double-mutant PB2 genes, each of which was rescued into an infectious virus. In general, the double-mutant PB2 transfectant viruses were more ts and attenuated in the lower respiratory tracts of hamsters than the single-mutant transfectant viruses, and the ts phenotype of two of three double-mutant PB2 transfectant viruses was stable even after prolonged replication in the upper respiratory tracts of immunocompromised mice. Two triple-mutant PB2 transfectant viruses with three predicted amino acid substitutions resulting from five nucleotide substitutions in the cDNA were then generated. The triple-mutant PB2 transfectant viruses were more ts and more attenuated than the double-mutant PB2 transfectant viruses. These results indicate that sequential introduction of additional ts mutations into the PB2 gene can yield mutants that exhibit a stepwise increase in temperature sensitivity and attenuation compared with the preceding mutant(s) in the series. Furthermore, the level of temperature sensitivity of the transfectant viruses correlated significantly with the level of attenuation of these viruses in hamsters. Although the triple-mutant PB2 transfectant viruses were attenuated in hamsters, intranasal administration of these viruses elicited a vigorous serum hemagglutination-inhibiting antibody response, and this was associated with resistance of the lower respiratory tract to subsequent wt virus challenge. These observations suggest the feasibility of using PB2 reverse genetics to generate a live influenza A virus vaccine donor strain that contains three attenuating mutations in one gene. It is predicted that reassortant viruses derived from such a donor virus would have the properties of attenuation, genetic stability, immunogenicity, and protective efficacy against challenge with wt virus.
Orthopoxvirus fusion inhibitor glycoprotein SPI-3 (open reading frame K2L) contains motifs characteristic of serine proteinase inhibitors that are not required for control of cell fusion
Authors: Turner P C , Moyer R W
Published: January 2020

The cowpox virus (CPV) SPI-3 gene (open reading frame K2L in vaccinia virus) is one of three orthopoxvirus genes whose products are members of the serpin (serine proteinase inhibitor) superfamily. The CPV SPI-3 gene, when overexpressed by using the vaccinia virus/T7 expression system, synthesized two proteins of 50 and 48 kDa. Treatment with the N glycosylation inhibitor tunicamycin converted the two SPI-3 proteins to a single 40-kDa protein, close to the size of 42 kDa predicted from the DNA sequence, suggesting that the SPI-3 protein, unlike the other two orthopoxvirus serpins, is a glycoprotein. Immunoblotting with an anti-SPI-3 antibody showed that the SPI-3 protein is synthesized early in infection prior to DNA replication. SPI-3 inhibits cell-cell fusion during infections with both CPV and vaccinia virus. A transfection assay was devised to test engineered mutants of SPI-3 for the ability to inhibit fusion. Two mutants with C-terminal deletions of 156 and 70 amino acids were completely inactive in fusion inhibition. Site-directed mutations were constructed near the C terminus of SPI-3, in or near the predicted reactive-site loop which is conserved in inhibitory serpins. Substitutions within the loop at the P1 to P1' positions and P5 to P5' positions, inclusive, did not result in any loss of activity, nor did changes at the P17 to P10 residues in the stalk of the reactive loop. Therefore, SPI-3 does not appear to control cell fusion by acting as a serine proteinase inhibitor.
The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing
Authors: Sandri-Goldin R M , Hibbard M K , Hardwicke M A
Published: January 2020

Herpes simplex virus type 1 infection results in a reorganization of antigens associated with the small nuclear ribonucleoprotein particles (snRNPs), resulting in the formation of prominent clusters near the nuclear periphery. In this study, we show that the immediate-early protein ICP27, which is involved in the impairment of host cell splicing and in the changes in the distribution of snRNPs, is also required for reassorting the SR domain splicing factor SC35. Other viral processes, such as adsorption and penetration, shutoff of host protein synthesis, early and late gene expression, and DNA replication, do not appear to play a role in changing the staining pattern of splicing antigens. Furthermore, the C-terminal repressor region of ICP27, which is required for the inhibitory effects on splicing, also is involved in redistributing the snRNPs and SC35. During infection or transfection with five different repressor mutants, the speckled staining pattern characteristic of uninfected cells was seen and the level of a spliced target mRNA was not reduced. Infections in the presence of activator mutants showed a redistributed snRNP pattern and a decreased accumulation of spliced target mRNA. Moreover, two arginine-rich regions in the N-terminal half of ICP27 were not required for the redistribution of snRNPs or SC35. Substitution of these regions with a lysine-rich sequence from simian virus 40 large-T antigen resulted in a redistribution of splicing antigens. Unexpectedly, a repressor mutant with a ts phenotype showed a redistributed staining pattern like that seen with wild-type infected cells. During infections with this ts mutant, splicing was not inhibited, as shown in this and previous studies, confirming its repressor phenotype. Furthermore, both the mutant and the wild-type protein colocalized with snRNPs. Therefore, the redistribution of snRNPs and SC35 correlates with ICP27-mediated impairment of host cell splicing, but these alterations are not sufficient to fully inhibit splicing. This indicates that active splicing complexes are still present even after dramatic changes in the organization of the snRNPs.
Induction of accelerated feline immunodeficiency virus disease by acute-phase virus passage
Authors: Diehl L J , Mathiason-Dubard C K , O'Neil L L , Obert L A , Hoover E A
Published: January 2020

Development of feline immunodeficiency virus (FIV) infection in cats as a small animal model for lentiviral immunodeficiency disease has been hampered by the prolonged and variable disease course following experimental infection. To address this issue, we generated high-titer, unselected FIV stocks by pooling plasma from cats acutely infected with a subgroup C FIV isolate designated CABCpadyOOC (FIV-C-PGammer). Subsequent infection with this virus pool resulted in rapidly progressive, fatal disease in greater than 50% of infected cats. Accelerated FIV disease was characterized by rapid and progressive CD4+ T-cell loss, lymphadenopathy, weight loss, lymphoid depletion, and severe thymic atrophy. Mortality and rate of disease progression were affected by the age of each cat at infection and whether the virus source animal was in the acute or chronic stage of infection. The rapid FIV disease syndrome was consistently associated with systemic lymphoid depletion, clinical disease, and susceptibility to opportunistic infections, analogous to accelerated and/or terminal HIV-1 infection. The results of this study demonstrate that FIV infection is a valid small animal model for lentiviral immunodeficiency disease.
High-level hepatitis B virus replication in transgenic mice
Authors: Guidotti L G , Matzke B , Schaller H , Chisari F V
Published: January 2020

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.
Gene targeting with a replication-defective adenovirus vector
Authors: Fujita A , Sakagami K , Kanegae Y , Saito I , Kobayashi I
Published: January 2020

Wide application of the gene-targeting technique has been hampered by its low level of efficiency. A replication-defective adenovirus vector was used for efficient delivery of donor DNA in order to bypass this problem. Homologous recombination was selected between a donor neo gene inserted in the adenovirus vector and a target mutant neo gene on a nuclear papillomavirus plasmid. These recombinant adenoviruses allowed gene transfer to 100% of the treated cells without impairing their viability. Homologous recombinants were obtained at a level of frequency much higher than that obtained by electroporation or a calcium phosphate procedure. The structure of the recombinants was analyzed in detail after recovery in an Escherichia coli strain. All of the recombinants examined had experienced a precise correction of the mutant neo gene. Some of them had a nonhomologous rearrangement of their sequences as well. One type of nonhomologous recombination took place at the end of the donor-target homology. The vector adenovirus DNA was inserted into some of the products obtained at a high multiplicity of infection. The insertion was at the end of the donor-target homology with a concomitant insertion of a 10-bp-long filler sequence in one of the recombinants. The possible relationship between these rearrangements and the homologous recombination is discussed. These results demonstrate the applicability of adenovirus-mediated gene delivery in gene targeting and gene therapy.
Anoxic induction of a sarcoma virus-related VL30 retrotransposon is mediated by a cis-acting element which binds hypoxia-inducible factor 1 and an anoxia-inducible factor
Authors: Estes S D , Stoler D L , Anderson G R
Published: January 2020

Cells exposed to hypoxia undergo substantial changes in gene expression generally associated with metabolic adaptation and increasing oxygen delivery. In contrast, responses distinct from those elicited by hypoxia are induced in anoxic fibroblasts; this includes activation of a set of VL30 elements. The responses seen in anoxically cultured fibroblasts are expressed physiologically in vivo during the anaerobic phase of wound healing. A fundamental question is whether transcriptional regulatory pathways utilized during anoxia are distinct from those already characterized for hypoxic cells. We report here the isolation of a 14-bp sequence within a VL30 retrotransposon promoter which mediates its anoxia responsiveness. Analyses of the protein complexes binding to this sequence demonstrated the presence of two distinct inducible DNA binding activities. The first is present in both hypoxic and anoxic fibroblasts and is indistinguishable from hypoxia-inducible factor 1. The second activity, which is present only in anoxic fibroblasts, is a previously uncharacterized heterodimeric DNA binding activity that appears to arise via posttranslational modification of an existing complex found in aerobic cells. These results indicate that the strong VL30 transcriptional induction seen with anoxia occurs through a mechanism specific to anoxia.
Retrovirus-like particles released from the human breast cancer cell line T47-D display type B- and C-related endogenous retroviral sequences
Authors: Seifarth W , Skladny H , Krieg-Schneider F , Reichert A , Hehlmann R , Leib-Mösch C
Published: January 2020

The human mammary carcinoma cell line T47-D releases retrovirus-like particles of type B morphology in a steroid-dependent manner (I. Keydar, T. Ohno, R. Nayak, R. Sweet, F. Simoni, F. Weiss, S. Karby, R. Mesa-Tejada, and S. Spiegelman, Proc. Natl. Acad. Sci. USA 81:4188-4192, 1984). Furthermore, reverse transcriptase (RT) activity is found to be associated with particle preparations. Using a set of degenerate primers derived from a conserved region of retroviral pol genes, we repeatedly amplified three different retroviral sequences (MLN, FRD, and FTD) from purified T47-D particles in several RT-PCR experiments. Screening of a human genomic library and Southern blot analysis revealed that these sequences are of endogenous origin. ERV-MLN represents a multicopy family of human endogenous retroviral elements (HERVs) with two closely related copies and up to 20 more distantly related members. In contrast, ERV-FRD and ERV-FTD comprise only one copy and five to seven related elements per haploid human genome. DNA sequence analysis of the proviral pol region of ERV-MLN revealed an uninterrupted stretch of 241 amino acids that shows 65% identity with the RT of the type B-related HERV designated HERV-K10. ERV-FRD and ERV-FTD are defective type C-related HERVs. The pol gene of ERV-FRD displays a nucleotide homology of 54% to the gibbon ape leukemia virus, and the pol gene of ERV-FTD is about 67% homologous to members of the RTVL-I family of HERVs. Our results thus indicate that the retroviral particles released by the breast cancer cell line T47-D are probably generated by complementation of several endogenous proviruses and can package retroviral transcripts of different origins.
A hepatitis A virus deletion mutant which lacks the first pyrimidine-rich tract of the 5' nontranslated RNA remains virulent in primates after direct intrahepatic nucleic acid transfection
Authors: Shaffer D R , Emerson S U , Murphy P C , Govindarajan S , Lemon S M
Published: January 2020

Cell culture-adapted variants of hepatitis A virus (HAV) in which the first pyrimidine-rich tract (pY1; nucleotides 99 to 138) of the 5' nontranslated region has been deleted (delta 96-137 or delta 96-139) replicate as well as parental virus in cultured cells (D.R. Shaffer, E.A. Brown, and S.M. Lemon, J. Virol. 68:5568-5578, 1994). To determine whether viruses with such large deletion mutations are able to replicate and to produce acute hepatitis in primates, we reconstructed the delta 96-137 deletion in the genetic background of a virulent virus which differs from the wild type by only one mutation in the 2B-coding region (HM175/8Y). Full-length synthetic delta 96-137/8Y RNA was injected into the livers of two HAV-seronegative marmosets (Saguinus mystax). Both animals developed serum liver enzyme elevations and inflammatory changes in serial liver biopsies within 3 to 4 weeks of inoculation which were comparable in magnitude to those observed previously following intrahepatic inoculation of marmosets with HM175/8Y RNA. Sequencing of RNA from virus shed in feces demonstrated the presence of the delta 96-137 deletion. These results indicate that the pY1 sequence of HAV is not required for efficient viral replication in hepatocytes in situ or for production of acute hepatic injury following intrahepatic RNA transfection in primates.
Rescue of vaccinia virus lacking the E3L gene by mutants of E3L
Authors: Chang H W , Uribe L H , Jacobs B L
Published: January 2020

Vaccinia virus with the E3L gene deleted was able to replicate in RK-13 but not HeLa cells. This host range phenotype could be complemented by an E3L gene expressed transiently from a plasmid. Analysis of mutants of E3L indicates that the ability to complement deletion of E3L correlates with the ability of mutated proteins to bind double-stranded RNA but not with their ability to migrate to the nucleus.