bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletions of the carboxy terminus of herpes simplex virus type 1 UL42 define a conserved amino-terminal functional domain
Authors: Tenney D J , Hurlburt W W , Bifano M , Stevens J T , Micheletti P A , Hamatake R K , Cordingley M G
Published: January 2020

The herpes simplex virus type 1 UL42 protein was synthesized in reticulocyte lysates and assayed for activity in vitro. Three functional assays were used to examine the properties of in vitro-synthesized UL42: (i) coimmunoprecipitation to detect stable complex formation with purified herpes simplex virus type 1 DNA polymerase (Pol), (ii) a simple gel-based assay for DNA binding, and (iii) a sensitive assay for the stimulation of Pol activity. UL42 synthesized in reticulocyte lysates formed a stable coimmunoprecipitable complex with Pol, bound to double-stranded DNA, and stimulated the activity of Pol in vitro. Carboxy-terminal truncations of the UL42 protein were synthesized from restriction enzyme-digested UL42 gene templates and gene templates made by polymerase chain reaction and assayed for in vitro activity. Truncations of the 488-amino-acid (aa) UL42 protein to aa 315 did not abolish its ability to bind to Pol and DNA or to stimulate Pol activity. Proteins terminating at aas 314 and 313 showed reduced levels of binding to Pol, but these and shorter proteins were unable to bind to DNA or to stimulate Pol activity. These results suggest that all three of the biochemical functions of UL42 colocalize entirely within the N-terminal 315 aas of the UL42 protein. Amino acid sequence alignment of alpha herpesvirus UL42 homologs revealed that the N-terminal functional domain corresponds to the most highly conserved region of the protein, while the dispensable C terminus is not conserved. Conservative aa changes at the C terminus of the 315-aa truncated protein were used to show that conserved residues were important for activity. These results suggest that 173 aa of UL42 can be deleted without a loss of activity and that DNA-binding and Pol-binding activities are correlated with the ability of UL42 to stimulate Pol activity.
Three baculovirus genes involved in late and very late gene expression: ie-1, ie-n, and lef-2
Authors: Passarelli A L , Miller L K
Published: January 2020

We developed a method to identify baculovirus genes required for late and very late gene expression that is based on subtraction of clones from an Autographa californica nuclear polyhedrosis virus genomic library which is able to trans activate promoters of reporter plasmids in transient expression assays. Using this assay, we found that three genes located between 83.7 and 7.5 map units of the Autographa californica nuclear polyhedrosis virus genome were involved in expression from the late capsid protein (vp39) and very late polyhedrin (polh) gene promoters. Two of these genes, ie-1 and ie-n, trans regulate early genes in transient expression assays. Although ie-1 was necessary and sufficient for expression from the early promoter in our assay, it was necessary but not sufficient for expression from the vp39 and polh promoters. The presence of ie-n increased expression from the early, late, and very late classes of promoters tested. However, a third gene identified in this region was specifically required for expression from the vp39 and polh promoters. This gene, a previously sequenced 630-nucleotide open reading frame, was renamed lef-2 for late expression factor 2. We also found that other genes in the region between 83.7 and 7.5 map units were not required for expression from the promoters used in this assay, although we did not eliminate the possibility that they subtly modify expression. These genes include pe-38 and me53, early genes with zinc finger-like motifs, and the upstream exon of ie-0, which specifies an alternate form of IE-1.
Analysis of transcription factors binding to the duplicated PEA1 and PEA3 sites that are required for polyomavirus mutant expression in PCC4 embryonic carcinoma cells
Authors: Nothias J Y , Weinmann R , Blangy D , Melin F
Published: January 2020

Embryonic carcinoma (EC) cell lines, representative of early embryonic undifferentiated cells, are nonpermissive for polyomavirus (PyV) infection as a result of a blockade of viral DNA early transcription and replication. All enhancers of PyV mutants (Py EC-PCC4), selected for the ability to grow on PCC4 EC cells, display a duplication of PEA1 and PEA3 binding sites (sites 1 and 3). However, the Py EC-PCC4 rearrangement is complex and results in variable mutant enhancer activities. We demonstrate here that duplication of sites 1 and 3 is absolutely required for a cooperative cis activation of early Py EC-PCC4 mutant transcription in PCC4 EC cells. In addition, we detect in PCC4 EC cells significant amounts of site 1- and 3-binding proteins, which we characterize as related to the Fos/Jun and Ets protein families, respectively. Wild-type PyV restriction in PCC4 EC cells may be relieved by a cooperation between site 2- and 3-binding proteins that would thereby be activated. Since site 1- or 3-binding factors could be derepressed, we improved the analysis of UV cross-linked DNA-protein complexes and were able to detect a novel factor, called PEA1/2 (for PyV enhancer A site 1- and 2-binding factor). Its DNA binding sequence overlaps sites 1 and 2 (PEA2 binding site) and is not duplicated in the M1 mutant, which exhibits the highest Py EC-PCC4 enhancer activity. he suggest that PEA1/2 is also involved in the regulation of PyV enhancer activity by repressing the site 1-binding activity.
Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase
Authors: Shibata S , Yamamoto-Goshima F , Maeno K , Hanaichi T , Fujita Y , Nakajima K , Imai M , Komatsu T , Sugiura S
Published: January 2020

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.
Mutational analysis of the leucine zipper-like motif of the human immunodeficiency virus type 1 envelope transmembrane glycoprotein
Authors: Chen S S , Lee C N , Lee W R , McIntosh K , Lee T H
Published: January 2020

The N-terminal region of the envelope (env) transmembrane protein of human immunodeficiency virus type 1 (HIV-1) has a leucine zipper-like motif. This highly conserved zipper motif, which consists of a heptad repeat of leucine or isoleucine residues, has been suggested to play a role in HIV-1 env glycoprotein oligomerization. This hypothesis was tested by replacing the highly conserved leucine or isoleucine residues in the zipper motif with a strong alpha-helix breaker, proline. We report here that such substitutions did not abolish the ability of env protein to form oligomers, indicating that this highly conserved zipper motif does not have a crucial role in env protein oligomerization. However, the mutant viruses all showed impaired infectivity, suggesting that this conserved zipper motif can have an important role in the virus life cycle.
Encapsidation of genetically engineered poliovirus minireplicons which express human immunodeficiency virus type 1 Gag and Pol proteins upon infection
Authors: Porter D C , Ansardi D C , Choi W S , Morrow C D
Published: January 2020

The use of recombinant viruses for the expression of a wide array of foreign proteins has become commonplace during the last few years. Recently, we have described the construction and characterization of chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes in which the gag and pol genes of HIV-1 have been substituted for the VP2 and VP3 capsid genes of the P1 capsid precursor region of poliovirus. Transfection of these RNAs into tissue culture cells results in replication of the RNA genome and expression of HIV-1-P1 fusion proteins (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Here we report on the encapsidation and amplification of the minireplicons to obtain sufficient quantities for biological characterization. To do this, HIV-1-poliovirus minireplicon genomes containing the gag or pol gene were transfected into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus capsid precursor protein, P1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). The chimeric minireplicons replicated and expressed the appropriate HIV-1-P1 fusion proteins as determined by immunoprecipitation with HIV-1-specific antibodies. The encapsidated genomes were isolated by ultracentrifugation. Reinfection of cells with the encapsidated chimeric RNA genomes resulted in expression of the HIV-1-Gag-P1 or HIV-1-Pol-P1 fusion protein. Serial passaging of the encapsidated chimeric HIV-1-poliovirus genomes was accomplished by coinfecting cells with the encapsidated minireplicons and VV-P1, resulting in stocks of the encapsidated minireplicons. Northern (RNA) blot analysis of passaged material revealed that no detectable deletions of the chimeric genomes occurred during 14 serial passages. Infection of cells by the encapsidated minireplicons was blocked by antipoliovirus antibodies. Coinfection of cells with encapsidated minireplicons and type 1 Sabin poliovirus resulted in encapsidation of the chimeric genomes by wild-type poliovirus as measured by immunoprecipitation of the HIV-1-P1 fusion proteins with HIV-1-specific antibodies. The results of this study demonstrate the encapsidation of poliovirus minireplicons which express foreign proteins and point to the future use of this system as a potential vaccine vector.
Guanylylation and adenylylation of the alpha regulatory proteins of herpes simplex virus require a viral beta or gamma function
Authors: Blaho J A , Mitchell C , Roizman B
Published: January 2020

Herpes simplex virus genes form several groups whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Most of the products of the first group, the alpha genes, appear to have regulatory functions. We report that the alpha proteins, infected cell proteins 4, 0, 22, and 27 of herpes simplex virus 1 and 4, 0, and 27 of herpes simplex virus 2, were labeled in the isolated nuclei of infected HeLa cells with [alpha-32P]GTP or [alpha-32P]ATP late in infection and that these proteins represent the largest group of virus-specific proteins labeled in this fashion. Studies with [2-3H]ATP, in which the label is in the purine ring, showed that a portion of the label in alpha proteins and in at least one other infected cell protein is due to nucleotidylylation. Analyses of the labeling reactions in nuclei of (i) cells infected with temperature-sensitive mutants at nonpermissive temperatures, (ii) cells infected with wild-type virus and harvested at different times postinfection, and (iii) cells treated with inhibitors of protein synthesis or of synthesis of viral DNA led to the conclusion that viral gene functions expressed after the synthesis of alpha proteins are required for the labeling of the alpha proteins with [alpha-32P]GTP. We conclude that several of the alpha proteins are extensively posttranslationally modified and that these modifications include nucleotidylylation.
Functional and immunologic characterization of human immunodeficiency virus type 1 envelope glycoproteins containing deletions of the major variable regions
Authors: Wyatt R , Sullivan N , Thali M , Repke H , Ho D , Robinson J , Posner M , Sodroski J
Published: January 2020

Deletions of the major variable regions (V1/V2, V3, and V4) of the human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein were created to study the role of these regions in function and antigenicity. Deletion of the V4 region disrupted processing of the envelope glycoprotein precursor. In contrast, the deletion of the V1/V2 and/or V3 regions yielded processed exterior envelope glycoproteins that retained the ability to interact with the gp41 transmembrane glycoprotein and the CD4 receptor. Shedding of the gp120 exterior glycoprotein by soluble CD4 was observed for the mutant with the V3 deletion but did not occur for the V1/V2-deleted mutant. None of the deletion mutants formed syncytia or supported virus entry. Importantly, the affinity of neutralizing antibodies directed against the CD4-binding region for the multimeric envelope glycoprotein complex was increased dramatically by the removal of both the V1/V2 and V3 structures. These results indicate that, in addition to playing essential roles in the induction of membrane fusion, the major variable regions mask conserved neutralization epitopes of the HIV-1 gp120 glycoprotein from antibodies. These results explain the temporal pattern associated with generation of HIV-1-neutralizing antibodies following infection and suggest stratagems for eliciting improved immune responses to conserved gp120 epitopes.
Translation of the hepatitis B virus P gene by ribosomal scanning as an alternative to internal initiation
Authors: Fouillot N , Tlouzeau S , Rossignol J M , Jean-Jean O
Published: January 2020

The hepatitis B virus (HBV) P gene which encodes the reverse transcriptase and other proteins required for replication is expressed on the bicistronic mRNA pregenome which also encodes the capsid protein in its first cistron. Recent results have suggested that the hepadnaviral P gene is translated by internal entry of ribosomes upstream from the P gene, in the overlapping C gene. Using a reporter gene fused to the HBV C or P gene, we demonstrate that the C sequence does not allow internal initiation of translation. Alternatively, our results support a model in which the HBV P gene is translated by ribosomes which scan from the capped extremity of the bicistronic mRNA pregenome. The mechanism by which the ribosomes scan past four AUGs before they initiate translation at the P AUG was analyzed. Our results show that these AUGs are skipped via two mechanisms: leaky scanning on AUGs in a weak or suboptimal initiation context and translation of an out-of-C-frame minicistron followed by reinitiation at P AUG. The minicistron translation allows ribosomes to bypass an AUG in a favorable context that would otherwise be used as a start codon for translation of a truncated capsid protein. Our results suggest that this elaborated scanning mechanism permits the coordinate expression of the HBV C and P genes on the viral bicistronic mRNA pregenome.
Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens
Authors: Ruediger R , Hentz M , Fait J , Mumby M , Walter G
Published: January 2020

Protein phosphatase 2A consists of three subunits, the catalytic subunit (C) and two regulatory subunits (A and B). The A subunit has a rod-like shape and consists of 15 nonidentical repeats. It binds the catalytic subunit through repeats 11 to 15 at the C terminus and the tumor antigens encoded by small DNA tumor viruses through overlapping but distinct regions at N-terminal repeats 2 to 8. A model of the A subunit was developed on the basis of the fact that uncharged or hydrophobic amino acids are conserved at eight defined positions within each repeat. Helical wheel projections suggested that each repeat can be arranged as two interacting amphipathic helixes connected by a short loop. Mutational analysis of the A subunit revealed that the proposed loops are important for binding of tumor antigens, the B subunit, and the C subunit. Native gel analysis of mutant A subunits synthesized in vitro demonstrated that the binding region for the B subunit, previously thought to include repeats 2 to 8, covers repeats 1 to 10 and that the B and C subunits cooperate in binding to the A subunit.
Differential role of long terminal repeat control elements for the regulation of basal and Tat-mediated transcription of the human immunodeficiency virus in stimulated and unstimulated primary human macrophages
Authors: Moses A V , Ibanez C , Gaynor R , Ghazal P , Nelson J A
Published: January 2020

Primary human macrophages induced to differentiate through contact with autologous activated nonadherent cells were used to investigate the transcriptional mechanisms involved in reactivation of human immunodeficiency virus (HIV) replication. Through transient transfection experiments with an HIV long terminal repeat (LTR)-chloramphenicol acetyltransferase reporter construct, we show that macrophage differentiation results in a 20-fold upregulation of basal LTR activity. To identify sequence elements responsive to the differentiation process, point mutations introduced into the LTR were tested in differentiated and undifferentiated macrophages. Several elements were identified as positive regulators of basal transcription. TATA, Sp1, and NF-kappa B binding sites were the most influential. The low-affinity site for LBP-1 (UBP-1) functioned as a negative regulator of LTR activity in undifferentiated macrophages, but this influence was lost upon differentiation. When tat was cotransfected into the expression system, the requirement for LTR elements identified as important for positive regulation of basal transcription remained in undifferentiated macrophages. Interestingly, however, the mutations in positive control elements which debilitated activity in undifferentiated macrophages had no effect on LTR activity in differentiated macrophages. Thus, it appears that while HIV-LTR activity is highly dependent on cellular transcription factors in undifferentiated cells, in differentiated macrophages the viral protein Tat confers pliability on the LTR and facilitates autonomy from absolute cellular control mechanisms. In vivo, release from either positive or negative regulation via cellular proteins may facilitate reactivation of HIV in macrophages.
Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains
Authors: Kayman S C , Wu Z , Revesz K , Chen H , Kopelman R , Pinter A
Published: January 2020

The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the human serum was only partially blocked by V3 peptide, indicating that this infected individual produced antibodies against epitopes in V3 that were expressed on the fusion glycoprotein but not by synthetic peptides. These data demonstrated that the chimeric glycoproteins described here effectively present native epitopes present in the V1/V2 and V3 domains of gp120 and provide efficient methods for detection of antibodies directed against native epitopes in these regions and for characterization of such epitopes.
A simian immunodeficiency virus envelope V3 cytotoxic T-lymphocyte epitope in rhesus monkeys and its restricting major histocompatibility complex class I molecule Mamu-A*02
Authors: Watanabe N , McAdam S N , Boyson J E , Piekarczyk M S , Yasutomi Y , Watkins D I , Letvin N L
Published: January 2020

The use of the simian immunodeficiency virus (SIV) macaque model for assessing human immunodeficiency virus vaccine strategies will be facilitated by the characterization of predominant SIV cytotoxic T-lymphocyte (CTL) epitopes and their restricting major histocompatibility complex (MHC) class I molecules in macaque species. We now define a rhesus monkey SIVmac CTL epitope in the third hypervariable region of the envelope glycoprotein of the virus. This epitope, YNLTMKCR, contains the first two amino acids of a cysteine-cysteine loop which is the SIVmac analog of the human immunodeficiency virus type 1 V3 loop. We also employed one-dimensional isoelectric focusing to characterize the MHC class I molecule of the rhesus monkey that binds this SIVmac envelope peptide fragment. Cloning and sequencing the cDNA encoding this rhesus monkey MHC class I molecule demonstrates that it is a newly described HLA-A homolog, Mamu-A*02. This viral CTL epitope and its restricting MHC class I molecule will facilitate the use of the SIVmac rhesus monkey model for studies of envelope-based vaccine strategies and for exploring AIDS immunopathogenesis.
Optimal lengths for DNAs encapsidated by Epstein-Barr virus
Authors: Bloss T A , Sugden B
Published: January 2020

We measured the efficiency of DNA packaging by Epstein-Barr virus (EBV) as a function of the length of the DNA being packaged. Plasmids that contain oriP (the origin of latent EBV DNA replication), oriLyt (the origin of lytic EBV DNA replication), the viral terminal repeats (necessary for cleavage and packaging by EBV), and various lengths of bacteriophage lambda DNA were introduced into EBV-positive cells. Upon induction of the resident EBV's lytic phase, introduced plasmids replicated as concatemers and were packaged. Plasmid-derived concatemers of DNA with certain lengths were found to predominate in isolated virion particles. We measured the distribution of lengths of plasmid concatemers found within cells supporting the lytic phase of the viral life cycle and found that this distribution differed from the distribution of lengths of concatemers found in mature virion particles. This finding indicates that the DNA packaged into mature virions represents a selected subset of those present in the cell during packaging. These observations together indicate that the length of DNA affects the efficiency with which that DNA is packaged by EBV. Finally, we measured the length of the packaged B95-8 viral DNA and found it to be approximately 165 kbp, or 10 kbp shorter than the originally predicted size for B95-8 based on its sequence. Together with the results of other studies, these findings indicate that the packaging of DNAs by EBV is dependent on two imprecisely recognized elements: the viral terminal repeats and the length of the DNA being packaged by the virus.
Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev response element decoy consisting of the 13-nucleotide minimal Rev-binding domain
Authors: Lee S W , Gallardo H F , Gilboa E , Smith C
Published: January 2020

Intracellular immunization is an anti-viral gene therapy strategy based on the introduction of DNA templates into cells to stably express genetic elements which inhibit viral gene expression and replication. We have recently developed an intracellular immunization strategy for human immunodeficiency virus (HIV) infection that uses RNA decoys. RNA decoys are short RNA oligonucleotides corresponding to the HIV trans activation response element (TAR) or Rev response element (RRE) sequences, which function by inhibiting the binding of the HIV regulatory proteins Tat and Rev to the authentic HIV RNA TAR and RRE regions, respectively. In this report we describe the characterization of potent RRE decoys containing the minimal 13-nucleotide primary Rev binding domain of the RRE. Using an improved tRNA cassette to express high levels of RRE transcripts in CEM cells, we found that this new generation of minimal RRE decoys were more potent inhibitors of HIV in isolated cell lines than previously described TAR or RRE decoys. CEM cells expressing RRE decoys exhibited diminished Rev function in cotransfection assays, confirming the specificity of inhibition of HIV by RRE decoys and indicating that the 13-nucleotide minimal Rev binding domain defined by using in vitro binding studies also binds Rev in vivo. Significant differences in the degree of HIV inhibition between individual CEM cell lines transduced with RRE decoy vectors which were not due to sequence alterations in the tRNA-RRE DNA template, differences in RRE decoy expression level, or endogenous variations in the resistance of CEM clonal cell lines to HIV were observed. In order to evaluate the efficacy of RRE decoys in a more realistic fashion than by comparison of individual clonal cell lines, polyclonal populations of transduced CEM cells were infected with HIV. By using a novel flow cytometric method for quantitating intracellular p24 expression, one version of the RRE decoys tested in this study was found to be capable of durably protecting polyclonal populations of CEM cells from HIV.
Redesignation of the RNase D activity associated with retroviral reverse transcriptase as RNase H
Authors: Hostomsky Z , Hughes S H , Goff S P , Le Grice S F
Published: January 2020

In the presence of Mn2+, reverse transcriptase of both human immunodeficiency virus and murine leukemia virus hydrolyzes duplex RNA. However, designating this novel activity RNase D conflicts with Escherichia coli RNase D, which participates in tRNA processing. On the basis of its location in the RNase H domain, we propose that this novel retroviral activity be redesignated RNase H*.
gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment
Authors: Marcon L , Sodroski J
Published: January 2020

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.
In vivo cooperation of two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, leads to transformation and immortalization of chicken myelomonocytic cells
Authors: Adelmant G , Quatannens B , Lagrou C , Wernert N , Torpier G , Saule S , Stehelin D , Laudet V
Published: January 2020

To investigate a possible in vivo cooperation between the p61/63myc and P135gag-myb-ets proteins, we used a previously constructed retrovirus, named MHE226, which contains the fused v-myb and v-ets oncogenes of the E26 retrovirus and the v-myc oncogene of MH2. For that purpose, chicken neuroretina cells producing MHE226 and pseudotyped with the Rous associated virus-1 (RAV-1) helper virus were injected in 1-day-old chickens. In control experiments, we also injected chicken neuroretina cells producing E26 (RAV-1), RAV-1 alone, or constructs lacking one of the oncogenes of MHE226. The average life span of MHE226-infected chickens is half that of E26-infected chickens. MHE226-infected chickens harbor tumors scattered in many organs, but compared with E26, MHE226 induced a weak leukemia. Study of integration sites suggests that the majority of the tumors results from clonal or oligoclonal events. Cell cultures were derived from the tumors of MHE226-infected chickens and grown in standard medium without addition of exogenous chicken myelomonocytic growth factor. These cells still divide at high rate after more than 100 passages and can thus be considered immortalized. By using several criteria, these cells were characterized as precursors of the myelomonocytic lineages.
Human immunodeficiency virus type 1 Rev-responsive element RNA binds to host cell-specific proteins
Authors: Shukla R R , Kimmel P L , Kumar A
Published: January 2020

RNase protection-gel retention studies show human host cell-specific ribonucleoprotein complexes with human immunodeficiency virus type 1 Rev-responsive element (RRE) RNA. Nuclear proteins from rodent or murine cells appear to lack the ability to form these complexes. Human-mouse somatic cell hybrids retaining a single human chromosome, either 6 or 12, form the RRE-nuclear-protein complexes. One of the complexes requires the entire RRE RNA, while the other needs RRE RNA stem-loops 1 and 2 only. Two major proteins with molecular masses of 120 and 62 kDa specifically bind to RRE RNA. Rodent cells (CHO) either lack or contain small amounts of these RRE-binding proteins.
Role of alveolar type II cells and of surfactant-associated protein C mRNA levels in the pathogenesis of respiratory distress in mink kits infected with Aleutian mink disease parvovirus
Authors: Viuff B , Aasted B , Alexandersen S
Published: January 2020

Neonatal mink kits infected with Aleutian mink disease parvovirus (ADV) develop an acute interstitial pneumonia with clinical symptoms and pathological lesions that resemble those seen in preterm human infants with respiratory distress syndrome and in human adults with adult respiratory distress syndrome. We have previously suggested that ADV replicates in the alveolar type II epithelial cells of the lung. By using double in situ hybridization, with the simultaneous use of a probe to detect ADV replication and a probe to demonstrate alveolar type II cells, we now confirm this hypothesis. Furthermore, Northern (RNA) blot hybridization showed that the infection caused a significant decrease of surfactant-associated protein C mRNA produced by the alveolar type II cells. We therefore suggest that the severe clinical symptoms and pathological changes characterized by hyaline membrane formation observed in ADV-infected mink kits are caused by a dysfunction of alveolar surfactant similar to that observed in respiratory distress syndrome in preterm infants. However, in the infected mink kits the dysfunction is due to the replication of ADV in the lungs, whereas the dysfunction of surfactant in preterm infants is due to lung immaturity.
A herpes simplex virus 1 US11-expressing cell line is resistant to herpes simplex virus infection at a step in viral entry mediated by glycoprotein D
Authors: Roller R J , Roizman B
Published: January 2020

A baby hamster kidney [BHK(tk-)] cell line (US11cl19) which stably expresses the US11 and alpha 4 genes of herpes simplex virus 1 strain F [HSV-1(F)] was found to be resistant to infection with HSV-1. Although wild-type HSV-1(F) attached with normal kinetics to the surface of US11cl19 cells, most cells showed no evidence of infection and failed to accumulate detectable amounts of alpha mRNAs. The relationship between the expression of UL11 and resistance to HSV infection in US11cl19 cells has not been defined, but the block to infection with wild-type HSV-1 was overcome by exposing cells with attached virus on their surface to the fusogen polyethylene glycol, suggesting that the block to infection preceded the fusion of viral and cellular membranes. An escape mutant of HSV-1(F), designated R5000, that forms plaques on US11cl19 cells was selected. This mutant was found to contain a mutation in the glycoprotein D (gD) coding sequence that results in the substitution of the serine at position 140 in the mature protein to asparagine. A recombinant virus, designated R5001, was constructed in which the wild-type gD gene was replaced with the R5000 gD gene. The recombinant formed plaques on US11cl19 cells with an efficiency comparable to that of the escape mutant R5000, suggesting that the mutation in gD determines the ability of the mutant R5000 to grow on US11cl19 cells. The observation that the US11cl19 cells were slightly more resistant to fusion by polyethylene glycol than parental BHK(tk-) cells led to the selection and testing of clonal lines from unselected and polyethylene glycol-selected BHK(tk-) cells. The results were that 16% of unselected to as much as 36% of the clones selected for relative resistance to polyethylene glycol fusion exhibited various degrees of resistance to infection. The exact step at which the infection was blocked is not known, but the results illustrate the ease of selection of cell clones with one or more sites at which infection could be blocked.
Cooperativity among herpes simplex virus type 1 immediate-early regulatory proteins: ICP4 and ICP27 affect the intracellular localization of ICP0
Authors: Zhu Z , Cai W , Schaffer P A
Published: January 2020

The results of transient expression assays and studies of viral mutants have shown that three of the five immediate-early proteins of herpes simplex virus type 1 (HSV-1) perform regulatory functions, individually and cooperatively. As part of efforts designed to explore the molecular basis for the functional cooperativity among ICP0, ICP4, and ICP27 in the regulation of HSV gene expression, we have examined the intracellular localization of ICP0 in cells infected with ICP4 and ICP27 null mutant viruses by indirect immunofluorescence. Although ICP0 was localized predominantly to the nuclei of wild-type virus-infected cells, it was found exclusively in the nuclei of ICP27 mutant-infected cells and in both the cytoplasm and nuclei of ICP4 mutant-infected cells, the cytoplasmic component being especially strong. These observations indicate that both ICP4 and ICP27 can affect the intracellular localization of ICP0. Transient expression assays with plasmids that express wild-type and mutant forms of ICP0, ICP4, and ICP27 confirmed that ICP4 promotes and that ICP27 inhibits the nuclear localization of ICP0. These results confirm the observations made for mutant virus-infected cells and indicate that the localization pattern seen in infected cells can be established by these three immediate-early proteins exclusive of other viral proteins. The C-terminal half of ICP27 was shown to be required to achieve its inhibitory effect on the nuclear localization of ICP0. The region of ICP0 responsive to ICP27 was mapped to the C terminus of the molecule between amino acid residues 720 and 769. In addition, the concentration of ICP27 was shown to have a significant effect on the intracellular localization of ICP0. Because the major regulatory activities of ICP0, ICP4, and ICP27 are expressed in the nucleus, the ability of these three proteins collectively to determine their own localization patterns within cells adds a new dimension to the complex process of viral gene regulation in HSV.
pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry
Authors: Ragheb J A , Anderson W F
Published: January 2020

Murine leukemia virus ecotropic and amphotropic envelope expression vectors were genetically engineered to generate truncations of the p15E TM cytoplasmic tail. The ecotropic construct CEET has the entire cytoplasmic tail of TM deleted, while the CEETR construct has only the R peptide portion of the tail deleted, thereby producing a TM subunit (p12E) that is identical to the one found in mature virions. The analogous amphotropic constructs were called CAET and CAETR. These envelopes, as opposed to their p15E TM counterparts, mediate cell-to-cell fusion at neutral pH in both transformed and nontransformed cell lines. Though the TM cytoplasmic domain is not required, its presence appears to augment such cell-to-cell fusion. This envelope-dependent fusion requires the presence of the viral receptor on the cell surface. Ecotropic virions bearing the p12E TM contain wild-type levels of the envelope complex and have near-normal titers. In contrast, virions which lack the cytoplasmic domain of TM (e.g., CEET) have 10- to 100-fold-lower titers but contain normal amounts of envelope. Both of the corresponding amphotropic virions contain normal amounts of envelope but have 10- to 100-fold-lower titers. Using immunofluorescent detection of envelope to monitor the fate of receptor-bound virions, we found that ecotropic murine leukemia virus envelope disappears from the cell surface while amphotropic envelope persists on the cell surface after virus binding. This pattern of immunofluorescence is consistent with the proposed routes of cell entry for these viruses, i.e., by endocytosis and direct fusion, respectively. In this assay, ecotropic virions bearing the genetically engineered p12E TM also appear to be internalized despite the ability of their envelope to mediate fusion at neutral pH in the same target cells. Our results show that direct fusion at neutral pH is a natural consequence of the surface expression of the mature ecotropic envelope and its receptor. We propose that the processing of the R peptide from the envelope TM (p15E) to yield p12E, at the time of virus budding or within virions, renders the envelope competent to fuse.
Construction of rubella virus genome-length cDNA clones and synthesis of infectious RNA transcripts
Authors: Wang C Y , Dominguez G , Frey T K
Published: January 2020

Plasmids containing a complete cDNA copy of the rubella virus (RUB) genomic RNA were constructed. Transfection into cell culture of genome-length RNA transcribed in vitro from one of these cDNA clones, Robo102, resulted in the production of virus which preserved the genetic and phenotypic characteristics of the parental virus from which the cDNA clone was derived. Prior to construction of the RUB genome-length cDNA clones, the 5'-terminal sequence of the RUB genomic RNA was determined to be 5'CAAUGG...3' following the cap structure. Analysis of the specific infectivity of RUB genomic RNA isolated from virions revealed that in Vero cells, the specific infectivity of RUB genomic RNA is roughly equivalent to that of Sindbis virus genomic RNA. In RUB virion RNA preparations, the subgenomic RNA was detected. It was demonstrated that subgenomic RNA was packaged into RUB virions; however, the presence of the subgenomic RNA was not essential for infectivity of the genomic RNA.
Biosynthesis and biochemical properties of the hepatitis C virus core protein
Authors: Santolini E , Migliaccio G , La Monica N
Published: January 2020

The biosynthesis and biochemical properties of the putative nucleocapsid protein of hepatitis C virus (HCV) were investigated. RNA transcripts for cell-free translation were prepared from truncated form of the cDNA construct encoding the structural proteins of HCV. Processing of the translation products was dependent on microsomal membranes and signal recognition particle, suggesting that release of the 21-kDa core protein from the polyprotein precursor is mediated solely by the signal peptidase of the endoplasmic reticulum (ER) and is achieved by the removal of a putative signal sequence of approximately 18 residues located at its C terminus. The core protein was found to bind membranes in vitro and in transfected cells, as shown by centrifugation analysis of in vitro translation products and transfected-cell lysates. Immunofluorescence of transfected cells showed that the core protein colocalized with the E2 glycoprotein as well as with a cellular ER membrane marker. The nucleocapsid protein expressed by in vitro translation in rabbit reticulocyte lysates cosedimented with the large ribosomal subunit in sucrose gradients. The ribosome binding domain was mapped to the N-terminal region of the core protein. Moreover, the same region was shown to bind RNA in vitro, suggesting that cosedimentation of core protein with ribosomes may be mediated by the RNA binding of the nucleocapsid protein of HCV. These studies indicate that the HCV core protein is a cytoplasmic protein associated with the ER membranes and possesses RNA binding activity.
Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription
Authors: Liao C L , Lai M M
Published: January 2020

We have developed a defective interfering (DI) RNA containing a chloramphenicol acetyltransferase reporter gene, placed behind an intergenic sequence, for studying subgenomic mRNA transcription of mouse hepatitis virus (MHV), a prototype coronavirus. Using this system, we have identified the sequence requirement for MHV subgenomic mRNA transcription. We show that this sequence requirement differs from that for RNA replication. In addition to the previously identified requirement for an intergenic (promoter) sequence, additional sequences from the 5' end of genomic RNA are required for subgenomic mRNA transcription. These upstream sequences include the leader RNA and a spacer sequence between the leader and intergenic sequence, which is derived from the 5' untranslated region and part of gene 1. The spacer sequence requirement is specific, since only the sequence derived from the 5' end of RNA genome, but not from other MHV genomic regions or heterologous sequences, could initiate subgenomic transcription from the intergenic sequence. These results strongly suggest that the wild-type viral subgenomic mRNAs (mRNA2 to mRNA7) and probably their counterpart subgenomic negative-sense RNAs cannot be utilized for mRNA amplification. Furthermore, we have demonstrated that a partial leader sequence present at the 5' end of genome, which lacks the leader-mRNA fusion sequence, could still support subgenomic mRNA transcription. In this case, the leader sequences of the subgenomic transcripts were derived exclusively from the wild-type helper virus, indicating that the MHV leader RNA initiates in trans subgenomic mRNA transcription. Thus, the leader sequence can enhance subgenomic transcription even when it cannot serve as a primer for mRNA synthesis. These results taken together suggest that the 5'-end leader sequence of MHV not only provides a trans-acting primer for mRNA initiation but also serves as a cis-acting element required for the transcription of subgenomic mRNAs. The identification of an upstream cis-acting element for MHV subgenomic mRNA synthesis defines a novel sequence requirement for regulating mRNA synthesis in RNA viruses.
Phenotypic mixing between different hepadnavirus nucleocapsid proteins reveals C protein dimerization to be cis preferential
Authors: Chang C , Zhou S , Ganem D , Standring D N
Published: January 2020

Hepadnaviruses encode a single core (C) protein which assembles into a nucleocapsid containing the polymerase (P) protein and pregenomic RNA during viral replication in hepatocytes. We examined the ability of heterologous hepadnavirus C proteins to cross-oligomerize. Using a two-hybrid assay in HepG2 cells, we observed cross-oligomerization among the core proteins from hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus. When expressed in Xenopus oocytes, in which hepadnavirus C proteins form capsids, the C polypeptides from woodchuck hepatitis virus and ground squirrel hepatitis virus, but not duck hepatitis B virus, can efficiently coassemble with an epitope-tagged HBV core polypeptide to form mixed capsids. However, when two different core mRNAs are coexpressed in oocytes the core monomers show a strong preference for forming homodimers rather than heterodimers. This holds true even for coexpression of two HBV C proteins differing only by an epitope tag, suggesting that core monomers are not free to diffuse and associate with other monomers. Thus, mixed capsids result from aggregation of different species of homodimers.