bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of translation initiation factor eIF-5A on the functioning of human T-cell leukemia virus type I Rex and human immunodeficiency virus Rev inhibited trans dominantly by a Rex mutant deficient in RNA binding
Authors: Katahira J , Ishizaki T , Sakai H , Adachi A , Yamamoto K , Shida H
Published: January 2020

The viral transactivator proteins Rex and Rev are necessary for the expression of structural proteins of human T-cell leukemia virus type I and human immunodeficiency virus type 1, respectively. Although the interaction of Rex/Rev with a cellular cofactor(s) has been thought to be required for Rex/Rev action, there is no suitable system to search for the cofactor(s) in mammalian cells. We found that a Rex mutant, TAgRex, which contains a simian virus 40 nuclear localization signal in place of the N-terminal 19 amino acids of Rex, could dominantly inhibit wild-type Rex/Rev functions. The inhibition did not require either Rev response element/Rex response element binding or the oligomerization ability of the mutant, but it did require a region around amino acid 90 of the Rex protein, suggesting that TAgRex sequestered the cellular cofactor. Complementation with the eukaryotic translation initiation factor 5A (eIF-5A) in this system could restore the impaired Rex function. These results indicate that eIF-5A is the cofactor indispensable for Rex function. Additionally, by using a two-hybrid system, the homo-oligomer formation of Rex was found to be mediated by the region around amino acid 90 in addition to Tyr-64 and Trp-65 of Rex protein. Thus, eIF-5A may play a part in the formation of the Rex homo-oligomer.
A replication-competent, endogenous retrovirus from an aged DBA/2 mouse contains the complete env from Emv-3 and a novel gag partially related to AKT-8
Authors: Bartman T , Murasko D M , Blank K J
Published: January 2020

We previously described an endogenous murine retrovirus, rv-DBA/2aged, isolated from an aged DBA/2 mouse. The previous report showed that a recombination which resulted in the replacement of Emv-3 gag sequences with gag sequences homologous to those found in the AKT-8 virus had taken place. This recombination allowed production of a competent virus from the defective Emv-3 locus. However, the extent of replacement of Emv-3 gag was not known. We report here the entire sequence for the gag gene of rv-DBA/2aged as well as the previously unsequenced 3' end of the Emv-3 gag gene. These data demonstrate that while sequences homologous to the entire gag gene fragment found in AKT-8 are represented in rv-DBA/2aged, the remainder of rv-DBA/2aged gag is not derived from Emv-3 but is a unique gag sequence. Furthermore, a complete comparison of env sequences shows that the env of rv-DBA/2aged is derived entirely from Emv-3. Additional data suggest that the recombination which led to production of the rv-DBA/2aged virus may be a common event in aging DBA/2 mice. Finally, comparison of the new sequences of Emv-3 with those of the Akv virus (also designated AKR-623 and Emv-11) and Emv-1 shows that this endogenous virus locus is very closely related to the other Emv loci at the nucleotide sequence level.
Brucella abortus conjugated with a gp120 or V3 loop peptide derived from human immunodeficiency virus (HIV) type 1 induces neutralizing anti-HIV antibodies, and the V3-B. abortus conjugate is effective even after CD4+ T-cell depletion
Authors: Golding B , Inman J , Highet P , Blackburn R , Manischewitz J , Blyveis N , Angus R D , Golding H
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) infection is associated with loss of function and numbers of CD4+ T-helper cells. In order to bypass the requirement for CD4+ cells in antibody responses, we have utilized heat-inactivated Brucella abortus as a carrier. In this study we coupled a 14-mer V3 loop peptide (V3), which is homologous to 9 of 11 amino acids from the V3 loop of HIV-1 MN, and gp120 from HIV-1 SF2 to B. abortus [gp120(SF2)-B. abortus]. Our results showed that specific antibody responses, dominated by immunoglobulin G2a in BALB/c mice, were induced by these conjugates. Sera from the immunized mice bound native gp120 expressed on the surfaces of cells infected with a recombinant vaccinia virus gp160 vector (VPE16). Sera from mice immunized with gp120(SF2)-B. abortus inhibited binding of soluble CD4 to gp120, whereas sera from mice immunized with V3-B. abortus were ineffective. Sera from mice immunized with either conjugate were capable of blocking syncytium formation between CD4+ CEM cells and H9 cells chronically infected with the homologous virus. Sera from mice immunized with gp120(SF2)-B. abortus were more potent than sera from mice immunized with V3-B. abortus in inhibiting syncytia from heterologous HIV-1 laboratory strains. Importantly, in primary and secondary responses, V3-B. abortus evoked anti-HIV MN antibodies in mice depleted of CD4+ cells, and sera from these mice were able to inhibit syncytia. These findings indicate that B. abortus can provide carrier function for peptides and proteins from HIV-1 and suggest that they could be used for immunization of individuals with compromised CD4+ T-cell function.
Nuclear export of the human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev is mediated by its activation domain and is blocked by transdominant negative mutants
Authors: Szilvay A M , Brokstad K A , Kopperud R , Haukenes G , Kalland K H
Published: January 2020

The human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev moves repeatedly between the cytoplasm, a perinuclear zone, the nucleoli, and nucleoplasmic speckles. In this study, we demonstrated by both indirect immunofluorescence and Western immunoblot analysis that nuclear exit of Rev transdominant negative mutants was defective compared with that of wild-type Rev. The basic and activation domains of Rev signal import and export, respectively, of Rev across the nuclear membrane. In cotransfection experiments, mutants containing mutations of Rev inhibited the nuclear egress of wild-type Rev, thus revealing a novel transdominant negative phenotype.
Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120
Authors: Matsushita S , Matsumi S , Yoshimura K , Morikita T , Murakami T , Takatsuki K
Published: January 2020

Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.
Identification and characterization of a cDNA derived from multiple splicing that encodes envelope glycoprotein gp105 of human herpesvirus 6
Authors: Pfeiffer B , Thomson B , Chandran B
Published: January 2020

The glycoprotein complex gp82-gp105 is a major virion envelope glycoprotein complex of human herpesvirus 6 variant A (HHV-6A) and consists of a number of related polypeptides. Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against this glycoprotein complex neutralized HHV-6A infectivity. We have previously reported the isolation, mapping, and characterization of a portion of the viral genomic DNA fragment encoding the gp82-gp105 complex and the identification of the neutralizing epitope (B. Pfeiffer, Z. N. Berneman, F. Neipel, C. K. Chang, S. Tirwatnapong, and B. Chandran, J. Virol. 67:4611-4620, 1993). This gene was further characterized by the identification of a 2.3-kb genomic fragment and by the identification of a 2.5-kb cDNA clone. The genomic sequence contains a short open reading frame (ORF) encoding the epitope recognized by the MAbs. The identified cDNA showed specificity for HHV-6 in Southern blot analysis with viral DNA. In Northern (RNA) blot analysis with total RNA from HHV-6A(GS)-infected cells, the cDNA insert specifically hybridized with several RNA species. Restriction mapping analysis localized this cDNA to the HHV-6A(U1102) genomic BamHI G fragment, at the right end of the unique long segment of the genome and to the SalI L and SalI O fragments within the left and right terminal direct repeat regions, respectively. In vitro transcription and translation of the cDNA revealed a polypeptide of about 88.5 kDa which was glycosylated in the presence of microsomal membranes to a polypeptide of approximately 104.2 kDa. Both polypeptides were immunoprecipiated by MAb 2D6, verifying the identity of the cDNA as encoding the gp105 in the gp82-gp105 complex. Sequence analysis of the cDNA revealed a large ORF potentially encoding a 650-amino-acid protein with 11 potential N-linked glycosylation sites and 18 cysteine residues. A potential membrane-spanning domain is located only near the amino terminus of the putative protein, indicating that gp105 may be a class 2 glycoprotein. Comparison of the cDNA nucleotide sequence with sequences from HHV-6A(U1102) genomic BamHI G and SalI L fragments revealed that the gene encoding gp105 contains 12 exons, spanning over 20 kb of the viral genome, with intron 1 spanning about 8 kb of genomic DNA. The first exon of the cDNA mapped to the right and left terminal direct repeats, while the other exons mapped within the unique long segment of the genome.(ABSTRACT TRUNCATED AT 400 WORDS)
Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59
Authors: Lu Y , Lu X , Denison M R
Published: January 2020

Gene 1 of the murine coronavirus, MHV-A59, encodes approximately 800 kDa of protein products within two overlapping open reading frames (ORFs 1a and 1b). The gene is expressed as a polyprotein that is processed into individual proteins, presumably by virus-encoded proteinases. ORF 1a has been predicted to encode proteins with similarity to viral and cellular proteinases, such as papain, and to the 3C proteinases of the picornaviruses (A. E. Gorbalenya, A. P. Donchenko, V. M. Blinov, and E. V. Koonin, FEBS Lett. 243:103-114, 1989; A. E. Gorbalenya, E. V. Koonin, A. P. Donchenko, and V. M. Blinov, Nucleic Acids Res. 17:4847-4861, 1989). We have cloned into a T7 transcription vector a cDNA fragment containing the putative 3C-like proteinase domain of MHV-A59, along with portions of the flanking hydrophobic domains. The construct was used to express a polypeptide in a combined in vitro transcription-translation system. Major polypeptides with molecular masses of 38 and 33 kDa were detected at early times, whereas polypeptides with molecular masses of 32 and 27 kDa were predominant after 30 to 45 min and appeared to be products of specific proteolysis of larger precursors. Mutations at the putative catalytic histidine and cysteine residues abolished the processing of the 27-kDa protein. Translation products of the pGpro construct were able to cleave the 27-kDa protein in trans from polypeptides expressed from the noncleaving histidine or cysteine mutants. The amino-terminal cleavage of the 27-kDa protein occurred at a glutamine-serine dipeptide as previously predicted. This study provides experimental confirmation that the coronaviruses express an active proteinase within the 3C-like proteinase domain of gene 1 ORF 1a and that this proteinase utilizes at least one canonical QS dipeptide as a cleavage site in vitro.
Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification
Authors: Verchot J , Carrington J C
Published: January 2020

The tobacco etch potyvirus (TEV) polyprotein is proteolytically processed by three viral proteinases (NIa, HC-Pro, and P1). While the NIa and HC-Pro proteinases each provide multiple functions essential for viral infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly. To determine if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire P1 coding region (delta P1 mutant) was produced with a modified TEV strain (TEV-GUS) expressing beta-glucuronidase (GUS) as a reporter, and its replication and movement phenotypes were assayed in tobacco protoplasts and plants. The delta P1 mutant accumulated in protoplasts to approximately 2 to 3% the level of parental TEV-GUS, indicating that the P1 protein may contribute to but is not strictly required for viral RNA amplification. The delta P1 mutant was capable of cell-to-cell and systemic (leaf-to-leaf) movement in plants but at reduced rates compared with parental virus. This is in contrast to the S256A mutant, which encodes a processing-defective P1 proteinase and which was nonviable in plants. Both delta P1 and S256A mutants were complemented by P1 proteinase expressed in a transgenic host. In transgenic protoplasts, genome amplification of the delta P1 mutant relative to parental virus was stimulated five- to sixfold. In transgenic plants, the level of accumulation of the delta P1 mutant was stimulated, although the rate of cell-to-cell movement was the same as in nontransgenic plants. Also, the S256A mutant was capable of replication and systemic infection in P1-expressing transgenic plants. These data suggest that, in addition to providing essential processing activity, the P1 proteinase functions in trans to stimulate genome amplification.
Growth kinetics of human cytomegalovirus are altered in monocyte-derived macrophages
Authors: Fish K N , Depto A S , Moses A V , Britt W , Nelson J A
Published: January 2020

Stimulation of monocytes/macrophages with activated nonadherent cells allows productive nonlytic growth of human cytomegalovirus (HCMV), but the viral replication cycle is delayed relative to replication of HCMV in human fibroblasts. Analysis of infected monocyte-derived macrophage (MDM) mRNA for major immediate-early (MIE 86, 72, and 55) and late (pp65 and gB) gene expression by reverse transcription PCR indicates that transcription peaks at 3 and 7 days postinfection (dpi), respectively. In contrast, in human fibroblast controls, mRNA for MIE and late gene expression peaked at 5 and 48 h postinfection, respectively. Consistent with reverse transcription PCR experiments, double-label antibody experiments first detected MIE antigen expression at 12 h postinfection, peaking at 3 dpi, and late (pp65 or gB) antigen expression at 5 dpi, peaking at 7 dpi. MIE antigen was not detected between 3 and 7 dpi but reappeared and was coexpressed with pp65 in enlarged MDM nuclei at 7 dpi. After 7 dpi, macrophages with numerous vacuoles containing large amounts of pp65 and gB were observed in culture. These vacuoles were frequently seen at cellular contact points, suggesting that cell-to-cell transfer of virus was the major mode of viral transmission. Consistent with this observation, infectious virus was recovered from MDM cellular lysates but not culture supernatant. The delayed growth and compartmentalization of HCMV in macrophages may allow the cell to accommodate the viral replication cycle without cell lysis. In addition, the macrophage may function as a vehicle for cell-to-cell transmission of HCMV.
Isolation of Epstein-Barr virus-infected clones of the human T-cell line MT-2: use of recombinant viruses with a positive selection marker
Authors: Fujiwara S , Ono Y
Published: January 2020

Certain classes of human T-cell lymphomas have been shown to be persistently infected with Epstein-Barr virus (EBV). To achieve an experimental system of persistent EBV infection in T cells, we used EBV recombinants with a positive selection marker. Infection of the human T-cell line MT-2 with EBV recombinants that had a hygromycin resistance gene and subsequent selection with this drug permitted isolation and long-term maintenance of EBV-infected MT-2 clones. For each clone, essentially 100% of cells were positive for EBV nuclear antigen. These MT-2 clones harbor monoclonal episomes of EBV DNA and stably express two EBV latent proteins, EBV nuclear antigen 1 and latent membrane protein 1. The growth of these EBV-infected MT-2 clones was slower than that of uninfected clones, suggesting that EBV affects regulation of T-cell proliferation. EBV recombinants with a positive selection marker will be a useful tool in establishing experimental systems of persistent EBV infection in certain non-B-cell lineages.
Characterization of cytotoxic cells from reovirus-infected SCID mice: activated cells express natural killer- and lymphokine-activated killer-like activity but fail to clear infection
Authors: Taterka J , Cebra J J , Rubin D H
Published: January 2020

Severe combined immune deficient (SCID) mice infected orally with reovirus type 1/L die of hepatitis. Leukocytes bearing the cell surface antigens Thy-1.2 and asialoGM-1 (AsGM1) accumulate in the livers of infected animals. These cells display lytic activity toward natural killer-sensitive (YAC-1) and -resistant (P815) cell lines and murine hepatoma line Hepa 1/A1. Although these cells have the capacity to lyse infected hepatoma targets, they cannot clear the virus.
Characterization of mouse parvovirus infection by in situ hybridization
Authors: Jacoby R O , Johnson E A , Ball-Goodrich L , Smith A L , McKisic M D
Published: January 2020

Infection of young adult BALB/cByJ mice with mouse parvovirus-1, a newly recognized, lymphocytotropic, nonpathogenic parvovirus, was examined by in situ hybridization. Virus appeared to enter through the small intestine and was disseminated to the liver and lymphoid tissues. Strand-specific probes detected virion DNA in a consistently larger number of cells than replicative forms of viral DNA and/or viral mRNA. The number of signal-positive cells in the intestinal mucosa, lymph nodes, spleen, and thymus increased through day 10 after oral inoculation but decreased after seroconversion. Positive cells were still detected, however, in peripheral lymphoid tissues of mice examined at 9 weeks postinoculation. The results underscore the need to assess potential effects of persistent mouse parvovirus-1 infection on immune function in mice.
The Epstein-Barr virus (EBV) BZLF2 gene product associates with the gH and gL homologs of EBV and carries an epitope critical to infection of B cells but not of epithelial cells
Authors: Li Q , Turk S M , Hutt-Fletcher L M
Published: January 2020

Glycoprotein gp85, the product of the BXLF2 open reading frame (ORF), is the gH homolog of Epstein-Barr virus (EBV) and has been implicated in penetration of virus into B cells. Like its counterparts in other herpesviruses, it associates with a gL homolog, gp25, which is the product of the BKRF2 ORF. Unlike the gH homologs of other herpesviruses, however, gp85 also complexes with two additional glycoproteins of 42 and 38 kDa. Glycoproteins gp42 and gp38 were determined to be alternatively processed forms of the BZLF2 gene product. Coexpression of EBV gH and gL facilitated transport of gH to the cell surface and resulted in formation of a stable complex of gH and gL. It also restored expression of an epitope recognized by monoclonal antibody E1D1, which immunoprecipitates the native gH complex but not recombinant gH expressed in isolation. Coexpression of gH, gL, and the BZLF2 ORF restored expression of an epitope recognized by a second monoclonal antibody, F-2-1, which immunoprecipitates the native gH-gL-gp42/38 complex but not the complex of recombinant gH and gL alone. The epitope recognized by antibody F-2-1 was mapped to the BZLF2 gene product itself. Antibody F-2-1 inhibited the ability of EBV to infect B lymphocytes but had no effect on the ability of the virus to infect the epithelial cell line SVK-CR2. In contrast, antibody E1D1 had no effect on infection of the B-cell line but inhibited infection of the epithelial cell line. These results indicate that penetration of the two cell types by EBV involves differential use of the gH-gL-gp42/38 complex and suggest the hypothesis that the BZLF2 gene product has evolved as a unique adaptation to infection of B lymphocytes by EBV.
Differential activation of the influenza virus polymerase via template RNA binding
Authors: Cianci C , Tiley L , Krystal M
Published: January 2020

Primary transcripts synthesized by the influenza virus polymerase contain the capped 5' ends of eukaryotic mRNAs. These sequences are derived from host mRNA and scavenged by the viral polymerase as a prerequisite to transcription. The first step in this reaction is the specific binding of the viral polymerase to the cap structure of the host RNA. The role that template RNA plays in this RNA binding reaction was examined in quantitative capped mRNA binding and endonuclease assays. Capped RNA binding was shown to be a template-dependent property of the influenza virus polymerase. Addition of only the 5' end of viral RNA stimulates capped mRNA binding by the viral polymerase, but endonuclease activity requires the addition of the 3' end. The addition of template RNA corresponding to the positive-sense complementary RNA replicative intermediate was also able to stimulate capped mRNA binding but was not able to efficiently activate the viral endonuclease. Thus, regulation of endonuclease activity by the influenza virus polymerase can be dependent on template RNA binding.
Illegitimate replication of linear hepadnavirus DNA through nonhomologous recombination
Authors: Yang W , Summers J
Published: January 2020

Linear hepadnavirus DNA in primary hepatocyte cultures efficiently participates in intra- and intermolecular nonhomologous recombination at its ends. The products of this recombination are (i) monomeric covalently closed circular DNAs (cccDNAs) with deletions and insertions around the site of joining and (ii) oligomeric forms in which monomers are joined near the ends in random orientation. A fraction of monomeric cccDNAs can serve as intermediates in further DNA replication through at least five generations of nonhomologous recombination in a process we call illegitimate replication. We suggest that the monomeric and oligomeric linear DNAs produced by illegitimate replication may be precursors of the integrated and other high-molecular-weight hepadnaviral DNA forms seen in chronic infection.
Transactivation activity of Meq, a Marek's disease herpesvirus bZIP protein persistently expressed in latently infected transformed T cells
Authors: Qian Z , Brunovskis P , Rauscher F , Lee L , Kung H J
Published: January 2020

Marek's disease virus (MDV) is an avian herpesvirus that induces a variety of diseases, including T-cell lymphomas, in chickens. In latently infected, transformed lymphoid cells, very few viral transcripts or proteins are detected. We previously described a gene, meq (MDV EcoQ), which is persistently expressed in MDV-transformed tumor samples and cell lines. meq codes for a 339-amino-acid protein with a basic-leucine zipper domain near its N terminus and a proline-rich domain near its C terminus. The basic-leucine zipper domain shows homology with Jun/Fos family proteins, whereas the proline-rich domain resembles that of the WT-1 tumor suppressor protein. These structural features raise the possibility that Meq functions as a transcription factor in regulating viral latency or oncogenesis. In this report, we show that the proline-rich domain is a potent transcription activator when fused to the yeast (Saccharomyces cerevisiae) Gal4(1-147) DNA-binding domain. The transactivation activity maps to the C-terminal 130 amino acids, with the last 33 amino acids essential. In the absence of these 33 amino acids, a two-and-one-half proline-rich repeat structure was found to exhibit repression activity. We further show that Meq is able to dimerize not only with itself but also with c-Jun. Meq/c-Jun heterodimers bind to an AP1-like sequence in the meq promoter region with an affinity much greater than that of Meq/Meq or c-Jun/c-Jun homodimers. Cotransfection chloramphenicol acetyltransferase assays suggest that the Meq/c-Jun heterodimers can up-regulate Meq expression in both chicken embryo fibroblasts and F9 cells. Our data provide the first biochemical evidence that Meq is a transcriptional factor and identify c-Jun as one of Meq's interacting partners.
Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell
Authors: Schwartz O , Maréchal V , Danos O , Heard J M
Published: January 2020

We have analyzed the replication of Nef+ and Nef- isogenic human immunodeficiency virus in CEM, HUT78, MT4 lymphoid, and U937 monocytic cell lines. At each passage of infected cells, we have assessed the relative infectivity of the virus particles released in culture media by measuring the number of infections units per nanogram of p24 protein. Values appeared to be 3- to 10-fold higher for the Nef+ virus than for the Nef- number The positive effect of Nef was observed regardless of the cell line, the multiplicity of infection, and the number of virus replication cycles achieved. We showed, by using cells expressing glycosylphosphatidylinositol-linked CD4, that the enhancement of virion infectivity could be dissociated from the down-regulation of cell surface CD4 also induced by Nef. The gp120-to-p24 ratio and the RNA content of virus particles produced in the presence or in the absence of Nef were equivalent. Virions bound to cell surface CD4 receptors with equal efficiencies. Equivalent reverse transcriptase activities were measured both on exogenous substrate and on particle genomic RNAs. In contrast, reverse transcription in infected cells generated 5- to 10-fold less DNA when the virions were produced in the absence of Nef, indicating that these particles performed reverse transcription in a suboptimal environment. These data suggest that the expression of Nef in virus-producing cells is required for efficient processing of the early stages of virus replication in target cells, including the internalization in an appropriate cell compartment and the uncoating of the particle.
Genetic analysis of the major homology region of the Rous sarcoma virus Gag protein
Authors: Craven R C , Leure-duPree A E , Weldon R A , Wills J W
Published: January 2020

The mature cores of all retroviruses contain a major structural protein known as the CA (capsid) protein. Although it appears to form a shell around the ribonucleoprotein complex that contains the viral RNA, its function in viral replication is largely unknown. Little sequence similarity exists between the CA proteins of different retroviruses, except for a region of about 20 amino acids termed the major homology region (MHR). To examine the role of the CA protein in particle assembly and release, mutants of Rous sarcoma virus were created in which segments of CA were deleted or single conserved residues in the MHR were altered. The ability of the deletion mutants to release particles at rates similar to the wild-type protein demonstrated that the CA domain of Gag is not an essential component of the minimal budding machinery. Certain point mutations in the MHR region did block assembly and release in certain cell types, presumably by perturbing the global structure of the Gag precursor. Another group of MHR substitutions produced noninfectious or poorly infectious particles that were normal in their content of gag and pol gene products and viral RNA. The mutants were capable of initiating reverse transcription in vitro; however, the association of CA protein with the core was compromised, as indicated by its sensitivity to extraction with nonionic detergent. Prominent blebs on the virion envelope also indicated a disturbance at the membrane. Finally, an anti-peptide serum directed against MHR was found to react with the uncleaved Gag protein but not with mature CA, suggesting that MHR undergoes a dynamic rearrangement upon liberation from the polyprotein. We conclude that the MHR is involved in the very late steps in maturation of the virion (i.e., ones that occur after budding is initiated) and is essential for proper function of the core upon entry into a new host cell.
Identification and characterization of the viral interaction determinant of the subgroup A avian leukosis virus receptor
Authors: Zingler K , Bélanger C a , Peters R , Agard E , Young J A
Published: January 2020

The cellular receptor for subgroup A avian leukosis viruses (ALV-A) has a small, 83-amino-acid extracellular domain containing a motif that is related in sequence to the ligand binding repeats of the low-density lipoprotein receptor. Extensive mutagenesis of the ALV-A receptor has identified two acidic amino acids (Asp-46 and Glu-47) and an adjacent aromatic amino acid (Trp-48) in the carboxy-terminal portion of this low-density lipoprotein receptor-related motif that are crucial for efficient viral entry. In addition, a 19-amino-acid peptide derived from this region efficiently and specifically blocked subgroup A viral infection when oxidized to form a disulfide bond previously predicted to form in the native receptor (C. Bélanger, K. Zingler, and J. A. T. Young, J. Virol. 69:1019-1024, 1995). Thus, the charged and aromatic amino acid determinants that are required for viral infection appear to lie on a small loop region of the ALV-A receptor. Previously, a single aromatic and one or more charged residues on the CD4 receptor for human and simian immunodeficiency viruses, and the MCAT receptor for ecotropic murine leukemia viruses, were shown to be important for viral entry. These results suggest that different retroviruses may recognize related determinants on structurally divergent cellular receptors.
The transcriptional regulatory proteins encoded by varicella-zoster virus open reading frames (ORFs) 4 and 63, but not ORF 61, are associated with purified virus particles
Authors: Kinchington P R , Bookey D , Turse S E
Published: January 2020

Of the five varicella-zoster virus (VZV) open reading frames (ORFs) known to encode proteins which influence viral transcriptional events, two (ORFs 10 and 62) encode proteins associated with the tegument of virus particles, where they may function during the immediate-early events of infection. In this study, antibodies which recognize the products of the three additional VZV ORFs, ORFs 4, 61, and 63, were made and used to characterize their association with virus particles. ORF 4 encoded a 52-kDa polypeptide, and antibodies to ORF 63 reacted with polypeptides of 47 and 28 kDa. Antibodies to ORF 61 recognized heterogeneous polypeptides of 62 to 66 kDa in cells infected with a vaccinia virus recombinant expressing ORF 61 and in VZV-infected melanoma cells but reacted very weakly with polypeptides of VZV-infected human foreskin fibroblasts, suggesting that cell-specific factors were involved in ORF 61 protein accumulation. Analysis of virus particles purified from melanoma cells indicated that a 52-kDa polypeptide from ORF 4 and the 47-kDa polypeptide from ORF 63, but not any from ORF 61, were associated with virus particles. The virion proteins were likely components of the tegument, as they were not solubilized by treatment of virus with mild detergents and were completely resistant to trypsin digestion unless prior envelope solubilization was performed. The products of ORFs 4 and 63 were not found in purified VZV nucleocapsids. These results suggest that forms of the ORF 4- and ORF 63-encoded transcriptional regulatory proteins are also structural and may also have roles in the immediate-early events of infection.
RNA sequences controlling the initiation and transfer of duck hepatitis B virus minus-strand DNA
Authors: Tavis J E , Ganem D
Published: January 2020

Hepadnaviruses replicate by reverse transcription of an RNA pregenome. Reverse transcription initiates within the stem-loop (SL) of the epsilon RNA packaging signal and is discontinuous: the nascent minus-polarity DNA is transferred to direct repeat 1 (DR1) at the 3' end of the pregenomic RNA prior to extensive elongation. In this study we analyzed the initiation and transfer of duck hepatitis B virus minus-strand DNA by using functional viral polymerase expressed in yeast cells. We extensively mutagenized both DR1 and the SL and observed the effects on reverse transcription initiation and on the transfer and subsequent extension of minus-strand DNA. Our results indicate that sequences throughout the SL affect initiation and that minus-strand DNAs initiated at three locations within the SL are competent for transfer to DR1. A short region of homology between the 5' end of minus-strand DNA and DR1 was necessary but not sufficient to direct the transfer and subsequent extension reactions. This homology was tolerant of minor substitutions, and 2 nucleotides of homology mediated transfer accurately. Mutations had greater detrimental effects on transfer and subsequent extension of minus-strand DNA when they were placed in DR1 than when they were placed in the SL. Efficient transfer of minus-strand DNA from a mutant SL to DR2 was observed in the yeast system. The hexanucleotide AAUUAC was identified as the primary cis element of the transfer acceptor, but this element was also insufficient to independently specify the acceptor location. Therefore, additional information, possibly positional context or unrecognized RNA secondary structure, is required.
Detection of CD4+ T cells harboring human immunodeficiency virus type 1 DNA by flow cytometry using simultaneous immunophenotyping and PCR-driven in situ hybridization: evidence of epitope masking of the CD4 cell surface molecule in vivo
Authors: Patterson B K , Goolsby C , Hodara V , Lohman K L , Wolinsky S M
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) infection of T cells and cells of the monocyte/macrophage lineage requires a specific interaction between the CD4 antigen expressed on the cell surface and the HIV-1 external envelope glycoprotein (gp120). To study the association between HIV-1 infection and modulation of cell surface expression of the CD4 molecule in vivo, we examined the CD4+ T cells harboring proviral DNA obtained from HIV-1-infected individuals who had received no antiretroviral therapy for at least 90 days. Simultaneous immunophenotyping of CD4 cell surface expression and PCR-driven in situ hybridization for HIV-1 DNA were used to resolve the CD4+ T cells into distinct populations predicted upon the presence or absence of proviral DNA. Among the HIV-1-infected study subjects, the percentage of CD4+ T cells harboring proviral DNA ranged from 17.3 to 55.5%, with a mean of 40.5%. Cell surface fluorescent staining with anti-CD4 antibody directed against a non-gp120 binding site-related epitope (L120) or a conformation-dependent epitope of the gp120 binding site (Leu 3A) demonstrated either an equivalent or a 1.5- to 3-fold-lower cell surface staining intensity for the HIV-1 DNA-positive subpopulation relative to the HIV-1 DNA-negative subpopulation, respectively. These data suggest that masking or alteration of specific epitopes on the CD4 molecule occurs after viral infection.
Effects on DNA synthesis and translocation caused by mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase
Authors: Blain S W , Goff S P
Published: January 2020

To determine the various roles of RNase H in reverse transcription, we generated a panel of mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase based on sequence alignments and the crystal structures of Escherichia coli and human immunodeficiency virus type 1 RNases H (S. W. Blain and S. P. Goff, J. Biol. Chem. 268:23585-23592, 1993). These mutations were introduced into a full-length provirus, and the resulting genomes were tested for infectivity by transient transfection assays or after generation of stable producer lines. Several of the mutant viruses replicated normally, some showed significant delays in infectivity, and others were noninfectious. Virions were collected, and the products of the endogenous reverse transcription reaction were examined to determine which steps might be affected by these mutations. Some mutants left their minus-strand strong-stop DNA in RNA-DNA hybrid form, in a manner similar to that of RNase H null mutants. Some mutants showed increased polymerase pausing. Others were impaired in first-strand translocation, independently of their wild-type ability to degrade genomic RNA, suggesting a new role for RNase H in strand transfer. DNA products synthesized in vivo by the wild-type and mutant viruses were also examined. Whereas wild-type virus did not accumulate detectable levels of minus-strand strong-stop DNA, several mutants were blocked in translocation and did accumulate this intermediate. These results suggest that in vivo wild-type virus normally translocates minus-strand strong-stop DNA efficiently.
Deletion of the baculovirus ecdysteroid UDP-glucosyltransferase gene induces early degeneration of Malpighian tubules in infected insects
Authors: Flipsen J T , Mans R M , Kleefsman A W , Knebel-Mörsdorf D , Vlak J M
Published: January 2020

Deletion of the ecdysteroid UDP-glucosyltransferase gene (egt) from the Autographa californica nuclear polyhedrosis virus (AcNPV) genome increases the speed of killing of this virus (D. R. O'Reilly and L. K. Miller, Bio/Technology 9:1086-1089, 1991). Second-instar Spodoptera exigua larvae are killed more rapidly by the egt deletion mutant of AcNPV than by wild-type AcNPV. Unlike wild-type AcNPV-infected larvae, larvae infected with an egt deletion mutant molt and resume feeding as mock-infected larvae do. Wild-type AcNPV and egt deletion mutant recombinants marked with a lacZ gene were used to study their pathogenesis in insects. Histopathological investigation revealed that early degeneration of the Malpighian tubules, not the molting per se, may be the cause of this increased speed of killing by AcNPV.
Expression of herpes simplex virus type 1 glycoprotein L (gL) in transfected mammalian cells: evidence that gL is not independently anchored to cell membranes
Authors: Dubin G , Jiang H
Published: January 2020

We expressed herpes simplex virus type 1 glycoprotein L (gL) in transfected cells to investigate whether it is independently anchored to plasma membranes or is membrane associated as a result of complex formation with gH. gL was detected by immunofluorescence microscopy at the surfaces of cotransfected cells when it was expressed with gH but not when it was expressed in the absence of gH or with a truncated form of gH, gHTrunc(792), which lacks the membrane-spanning region and terminates at amino acid 792. Immunoprecipitation studies of transfected-cell culture media revealed that gL was secreted from cells when expressed in the absence of gH and was secreted from cotransfected cells complexed with gHTrunc(792). These observations demonstrate that gL is not independently anchored to plasma membranes but is membrane associated as a result of complex formation with gH.
Long-term expression in sensory neurons in tissue culture from herpes simplex virus type 1 (HSV-1) promoters in an HSV-1-derived vector
Authors: Smith R L , Geller A I , Escudero K W , Wilcox C L
Published: January 2020

Amplicons, defective herpes simplex virus type 1 (HSV-1) vectors, were constructed to use four HSV-1 promoters, from the immediate-early (IE) 1 IE 3, IE 4/5, and late glycoprotein C (gC) genes, to regulate expression of the Escherichia coli lacZ gene, encoding beta-galactosidase, and packaged into infectious particles. Infection of sensory neurons in vitro with amplicons containing the IE 1, IE 3, or IE 4/5 promoter resulted in stable long-term expression of beta-galactosidase from 2 to 10 weeks after gene transfer. The number of neurons expressing beta-galactosidase was not changed by treatments previously shown to produce reactivation of latent HSV-1. In addition, the latency-associated transcript was detected in many of the same neurons that expressed beta-galactosidase, indicating that the viral IE promoters in the amplicons can function in the same neurons that harbor latent virus. Delivery of beta-galactosidase protein directly into neurons by microinjection indicated that the half-life for histochemical detection of beta-galactosidase was between 24 and 48 h, suggesting that the persistence of beta-galactosidase histochemical staining cannot be explained by the stability of the reporter protein alone. In contrast to the IE promoters, the gC promoter of the late gene class did not support long-term expression of beta-galactosidase; instead, beta-galactosidase was detected in only a few neurons per culture at 2 weeks after infection, and superinfection with wild-type HSV-1 did not increase the level of expression from the gC promoter. These results suggest that the HSV-1 IE promoters in the amplicons are not subject to the promoter inactivation that occurs with many types of virus vectors and that the IE promoters in the context of the amplicon avoid the promoter inactivation observed from the same promoters in the HSV-1 genome during latency.
Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression
Authors: Anton M , Graham F L
Published: January 2020

We have constructed replication-defective human adenovirus (Ad) type 5 vectors containing the gene for the Cre recombinase from bacteriophage P1 under control of the human cytomegalovirus immediate-early promoter (AdCre). Expression of the protein was detected in replication-permissive (293) and in nonpermissive (MRC5) cell lines, and its biochemical activity was demonstrated in a cell-free recombination assay using a plasmid containing two loxP sites. To study Cre-mediated recombination in an intracellular system, we constructed an Ad vector (AdMA19) containing the luciferase cDNA under control of the human cytomegalovirus promoter but separated from it by an extraneous spacer sequence flanked by loxP sites which blocked luciferase expression. Upon coinfection of 293 or MRC5 cells with AdMA19 and AdCre, luciferase expression was specifically induced by Cre-mediated excision of the intervening sequence. The use of Ad vectors combined with the Cre-loxP system for regulation of gene expression and other possible applications is discussed.
The cardiovirulent phenotype of coxsackievirus B3 is determined at a single site in the genomic 5' nontranslated region
Authors: Tu Z , Chapman N M , Hufnagel G , Tracy S , Romero J R , Barry W H , Zhao L , Currey K , Shapiro B
Published: January 2020

We report the construction of chimeric coxsackievirus B3 (CVB3) strains in which sequences of an infectious cDNA copy of a noncardiovirulent CVB3 genome were replaced by the homologous sequences from a cardiovirulent CVB3 genome to identify which of 10 predicted genetic sites determine cardiovirulence. Cardiovirulent phenotype expression was consistently linked to nucleotide 234 (U in cardiovirulent CVB3 and C in avirulent CVB3) in the 5' nontranslated region. Reconstructions of the parental noncardiovirulent CVB3 genome from chimeras restored the noncardiovirulent phenotype when tested in mice. Inoculation of severe combined immunodeficient (scid) mice with the noncardiovirulent CVB3 strain resulted in massive cardiomyocyte necrosis in all animals. Sequence analysis of viral genomes isolated from twelve scid mouse hearts showed that only nucleotide position 234 was different (a C-->U transition) from that in the input parental noncardiovirulent CVB3 genome. Higher-order RNA structures predicted by two different algorithms did not demonstrate an obvious local effect caused by the C-->U change at nucleotide 234. Initial studies of parental and chimeric CVB3 replication in primary cultures of fetal murine heart fibroblasts and in adult murine cardiac myocytes demonstrated that viral RNA transcriptional efficiency is approximately 10-fold lower for noncardiovirulent CVB3 than for cardiovirulent CVB3. CVB3 did not shut off protein synthesis in murine cardiac fibroblasts, nor were levels of viral protein synthesis significantly different as a function of viral phenotype. Taken together, these data support a significant role for determination of the CVB3 cardiovirulence phenotype by nucleotide 234 in the 5' nontranslated region, possibly via a transcriptional mechanism.
Vaccinia, cowpox, and camelpox viruses encode soluble gamma interferon receptors with novel broad species specificity
Authors: Alcamí A , Smith G L
Published: January 2020

Soluble receptors for gamma interferon (IFN-gamma) are secreted from cells infected by 17 orthopoxviruses, including vaccinia, cowpox, rabbitpox, buffalopox, elephantpox, and camelpox viruses, representing three species (vaccinia, cowpox, and campelpox viruses). The B8R open reading frame of vaccinia virus strain Western Reserve, which has sequence similarity to the extracellular binding domain of cellular IFN-gamma receptors (IFN-gamma Rs), is shown to encode an IFN-gamma binding activity by expression in recombinant baculovirus. The soluble virus IFN-gamma Rs bind IFN-gamma and, by preventing its interaction with the cellular receptor, interfere with the antiviral effects induced by this cytokine. Interestingly, in contrast to cellular IFN-gamma Rs, which are highly species specific, the vaccinia, cowpox, and camelpox virus IFN-gamma Rs bind and inhibit the biological activity of human, bovine, and rat IFN-gamma but not mouse IFN-gamma. This unique broad species specificity of the IFN-gamma R would aid virus replication in different species and suggests that vaccinia, cowpox, and camelpox viruses may have evolved in several species, possibly including humans but excluding mice. Last, the conservation of an IFN-gamma R in orthopoxviruses emphasizes the importance of IFN-gamma in defense against poxvirus infections.