bluuebunny/crossref_metadata_2025 huggingface dataset:
Kaposi's sarcoma-associated herpesvirus contains G protein-coupled receptor and cyclin D homologs which are expressed in Kaposi's sarcoma and malignant lymphoma
Authors: Cesarman E , Nador R G , Bai F , Bohenzky R A , Russo J J , Moore P S , Chang Y , Knowles D M
Published: January 2020

A new human herpesvirus was recently identified in all forms of Kaposi's sarcoma (Kaposi's sarcoma-associated herpesvirus [KSHV] or human herpesvirus 8), as well as in primary effusion (body cavity-based) lymphomas (PELs). A 12.3-kb-long KSHV clone was obtained from a PEL genomic library. Sequencing of this clone revealed extensive homology and colinearity with the right end of the herpesvirus saimiri (HVS) genome and more limited homology to the left end of the Epstein-Barr virus genome. Four open reading frames (ORFs) were sequenced and characterized; these are homologous to the following viral and/or cellular genes: (i) Epstein-Barr virus membrane antigen p140 and HVS p160, (ii) HVS and cellular type D cyclins, (iii) HVS and cellular G protein-coupled receptors, and (iv) HVS. Since there is considerable evidence that cyclin D1 and some G protein-coupled receptors contribute to the development of specific cancers, the presence of KSHV homologs of these genes provides support for a role for KSHV in malignant transformation. All ORFs identified are transcribed in PELs and Kaposi's sarcoma tissues, further suggesting an active role for KSHV in these diseases.
Previously unsuspected cis-acting sequences for DNA replication revealed by characterization of a chimeric heron/duck hepatitis B virus
Authors: Mueller-Hill K , Loeb D D
Published: January 2020

Heron hepatitis B virus (HHBV) is an avian hepadnavirus that is closely related to duck hepatitis B virus (DHBV). To learn more about the mechanism of hepadnavirus replication, we characterized a clone of HHBV that contains a substitution of DHBV sequence from nucleotide coordinates 403 to 1364. This clone, named HDE1, expresses a chimeric pregenomic RNA, a chimeric polymerase (P) protein, and a core (C) protein with a one-amino-acid substitution at its carboxy terminus. We have shown that HDE1 is defective for minus-strand DNA synthesis, resulting in an overall reduction of viral DNA. HDE1 was also defective for plus-strand DNA synthesis, resulting in aberrant ratios of replication intermediates. Genetic complementation assays indicated that HDE1 replication proteins, C and P, are functional for replication and wild-type HHBV proteins do not rescue either defect. These findings indicate that the HDE1 substitution mutation acts primarily in cis. By restoring nucleotides 403 to 902 to the HHBV sequence, we showed that cis-acting sequences for plus-strand DNA synthesis are located in the 5' half of the HDE1 chimeric region. These data indicate the presence of one or more formerly unrecognized cis-acting sequences for DNA synthesis within the chimeric region (nucleotides 403 to 1364). These cis-acting sequences in the middle of the genome might interact directly or indirectly with known cis elements that are located near the ends of the genome. Our findings suggest that a specific higher-order template structure is involved in the mechanism of hepadnavirus DNA replication.
Accessory human cytomegalovirus glycoprotein US9 in the unique short component of the viral genome promotes cell-to-cell transmission of virus in polarized epithelial cells
Authors: Maidji E , Tugizov S , Jones T , Zheng Z , Pereira L
Published: January 2020

Human cytomegalovirus (CMV) encodes accessory glycoproteins that are dispensable for virus growth in nonpolarized cells in culture. We report that CMV deletion mutants lacking the gene for accessory glycoprotein US9 in the unique short component of the viral genome are impaired in plaque formation in polarized human retinal pigment epithelial (ARPE-19) cells. Comparison of CMV deletion mutants in US9 with herpes simplex virus type 1 deletion mutants lacking glycoproteins gE and gI showed that both of these mutants are impaired in altering junctional complexes and increasing paracellular permeability in polarized ARPE-19 cells cultured on permeable filter supports. Results of functional studies indicate that CMV US9 and homologs of gE have analogous roles in promoting virus spread across lateral membranes of polarized epithelial cells.
A hybrid herpesvirus infectious vector based on Epstein-Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo
Authors: Wang S , Vos J M
Published: January 2020

We have developed a miniviral vector, pH300, based on the human herpesviruses 1 and 4, herpes simplex virus type 1 (HSV-1), and Epstein-Barr virus (EBV), carrying EBV sequences for plasmid episomal maintenance and HSV-1 sequences for amplification and packaging in multimeric form into HSV-1 capsids in the presence of a helper virus and helper cell line. A reporter gene, the bacterial lacZ gene, which expressed beta-galactosidase, was inserted into the multiple cloning site of pH300 to make pH300-lac. The packaged pH300-lac DNA was very efficient in infecting human cells in tissue culture. The pH300-lac miniviral stock was used to infect in vitro various human cell types derived from breast cancer, lung cancer, and liver cancer. Up to 95% of cells were infected and expressed beta-galactosidase activity after exposure to viral stock at a multiplicity of infection of 3. There was essentially no apparent cytotoxicity after infection of cultured cells in vitro. To test in vivo gene delivery, human liver tumor cells preimplanted subcutaneously in nude mice and injected in situ with pH300-lac showed high efficiency of ectopic gene expression. The pH300 miniviral vector is a simple and effective gene transfer system which shows potential for gene therapy of cancer and inherited diseases.
Intermediate class of mRNAs in African swine fever virus
Authors: Rodríguez J M , Salas M L , Viñuela E
Published: January 2020

A transcriptional analysis of the African swine fever virus (ASFV) I226R and I243L genes is presented. Steady-state kinetics and transfection experiments showed the existence of a new temporal class of ASFV mRNAs transcribed from these genes, with the characteristics of the poxvirus intermediate transcripts. Transcription of the I226R gene gave rise to intermediate and late mRNAs that started from different sites, while the I243L gene produced early, intermediate, and late mRNAs, also starting from different sites. The presence of intermediate genes suggests a cascade model for the regulation of ASFV gene expression.
Human cytomegalovirus mtrII oncoprotein binds to p53 and down-regulates p53-activated transcription
Authors: Muralidhar S , Doniger J , Mendelson E , Araujo J C , Kashanchi F , Azumi N , Brady J N , Rosenthal L J
Published: January 2020

The 79-amino-acid (79-aa) open reading frame (UL111a) gene within morphological transforming region II (mtrII) of human cytomegalovirus strain Towne has been shown to transform rodent cells in vitro (J. Thompson, J. Doniger, and L. J. Rosenthal, Arch. Virol. 136:161-172, 1994). Moreover, a translation termination linker (TTL) mutant of mtrII that coded for the first 49 aa of mtrII oncoprotein (designated TTL49) was sufficient for malignant transformation, whereas a TTL mutant that coded for the first 24 aa (designated TTL24) was not. The current study demonstrates the binding of mtrII oncoprotein to the tumor suppressor protein p53 both in vivo using transiently transfected cells and in vitro using labeled proteins. Furthermore, the C-terminally truncated mtrII protein TTL49, but not truncated protein TTL24, bound to p53. The mtrII binding domain mapped to the N-terminal region of p53, residues 1 to 106, with a critical region from aa 27 to 44, whereas the p53 binding domain of mtrII protein was the first 49 aa. Furthermore, mtrII inhibited p53-activated transcription, indicating its ability to alter p53-directed cellular regulatory mechanisms. mtrII oncoprotein was detected both in stably transfected NIH 3T3 cell lines and human cytomegalovirus-infected HEL 299 cells (as early as 12 h after infection) in the perinuclear region and in the nucleus. mtrII-transformed cell lines, at both early and late passage, exhibited high levels of p53 with a 15-fold-extended half-life. However, p53-activated transcription was suppressed in these cells in spite of the increased p53 levels. Finally, the results with wild-type mtrII and its TTL mutants with respect to p53 binding, p53-activated transcription, and transforming ability suggest that the mechanism of mtrII transformation is linked to both p53 binding and disruption of p53 cell regulation.
cis- and trans-acting elements involved in reactivation of vaccinia virus early transcription
Authors: Masternak K , Wittek R
Published: January 2020

We have previously shown that transcription from the vaccinia virus 7.5K early promoter is reactivated late in infection (J. Garcés, K. Masternak, B. Kunz, and R. Wittek, J. Virol. 67:5394-5401, 1993). To identify the sequence elements mediating reactivation, we constructed recombinant viruses harboring deletions, substitutions, or insertions in the 7.5K promoter or its flanking regions. The analysis of these viruses showed that sequences both upstream as well as downstream of the transcription initiation site contribute to reactivation of the 7.5K promoter. We tested whether reactivation could be explained by a high affinity of vaccinia virus early transcription factor to reactivated promoters. Bandshift experiments using purified protein showed that promoters which bind the factor with high affinity in general also have high early transcriptional activity. However, no correlation was found between affinity of the factor and reactivation. Interestingly, overexpression of recombinant early transcription factor in vaccinia virus-infected cells resulted in a shutdown of late transcription and in reactivation of promoters, which are normally not reactivated.
A specific subform of the human cytomegalovirus transactivator protein pUL69 is contained within the tegument of virus particles
Authors: Winkler M , Stamminger T
Published: January 2020

The polypeptide encoded by the open reading frame UL69 of human cytomegalovirus (HCMV), which is homologous to the immediate-early regulator ICP27 of herpes simplex virus, has recently been identified as a transactivator protein that exerts a broad stimulatory effect on gene expression (M. Winkler, S. A. Rice, and T. Stamminger, J. Virol. 68:3943-3954, 1994). Here, we provide evidence that pUL69 is a phosphorylated tegument protein of HCMV. This finding could be demonstrated by Western blot (immunoblot) analyses with purified virions and a specific antiserum against pUL69. These experiments revealed that one phosphorylated subform of the three pUL69 polypeptides that are synthesized in infected fibroblast cells is contained within the HCMV virion. After the treatment of purified virions with detergents, pUL69 could not be detected within the membrane fraction, suggesting that it is either a capsid or a tegument protein. Its presence within dense bodies, however, shows that pUL69 is a constituent of the viral tegument.
T-cell-mediated immunity to lymphocytic choriomeningitis virus in beta2-integrin (CD18)- and ICAM-1 (CD54)-deficient mice
Authors: Christensen J P , Marker O , Thomsen A R
Published: January 2020

The T-cell response to lymphocytic choriomeningitis virus was studied in mice with deficient expression of beta2-integrins or ICAM-1. In such mice, the generation of virus-specific cytotoxic T lymphocytes was only slightly impaired and bystander activation was as extensive as that observed in wild-type mice. T-cell-mediated inflammation, assessed as primary footpad swelling and susceptibility to intracerebral infection, was slightly compromised only in beta2-integrin-deficient mice. However, adoptive immunization of mutant mice soon after local infection did reveal a reduced capacity to support the inflammatory reaction, indicating that under conditions of more limited immune activation both molecules do play a role in formation of the inflammatory exudate. Finally, virus control was found to be somewhat impaired in both mutant strains. In conclusion, our results indicate that although LFA-1-ICAM-1 interaction is important for certain aspects of the T-cell-mediated response to viruses, T-cell activation is surprisingly intact in these mutant mice, indicating extensive functional redundancy within cell interaction molecules.
Role of different lymphoid tissues in the initiation and maintenance of DNA-raised antibody responses to the influenza virus H1 glycoprotein
Authors: Boyle C M , Morin M , Webster R G , Robinson H L
Published: January 2020

Antibody responses in mice immunized by a single gene gun inoculation of plasmid expressing the influenza virus H1 hemagglutinin and in mice immunized by a sublethal H1 influenza virus infection have been compared. Both immunizations raised long-lived serum responses that were associated with the localization of antibody-secreting cells (ASC) to the bone marrow. However, the kinetics of these responses were 4 to 8 weeks slower in the DNA-immunized than in the infection-primed mice. Following a gene gun booster, the presence of ASC in the inguinal lymph nodes, but not in other lymph nodes, revealed gene gun responses being initiated in the nodes that drain the skin target site. Both pre- and postchallenge, the DNA-immunized mice had 5- to 10-times-lower levels of antibody and ASC than the infection-primed mice.
Role of protein kinase A and the serine-rich region of herpes simplex virus type 1 ICP4 in viral replication
Authors: Xia K , Knipe D M , DeLuca N A
Published: January 2020

Efficient expression of herpes simplex virus genes requires the synthesis of functional ICP4, a nuclear phosphoprotein that contains a prominent serine-rich region between amino acids 142 and 210. Residues in this region not only are potential sites for phosphorylation but also are involved in the functions of ICP4. By comparing the growth of a virus in which this region is deleted (d8-10) with wild-type virus (KOS) in PC12 cells or PC12 cells that are deficient in cyclic AMP-dependent protein kinase (PKA), two observations were made: (i) the growth of wild-type virus was impaired by 1 to 2 orders of magnitude in the PKA-deficient cells, indicating the involvement of PKA in the growth cycle of herpes simplex virus type 1, and (ii) while the growth of d8-10 was impaired by almost 2 orders of magnitude in wild-type cells, it was not further impaired (as was that of wild-type virus) in PKA-deficient cells, implicating the region deleted in d8-10 as a possible target for cellular PKA. In trigeminal'ganglia of mice, the d8-10 mutant virus grew poorly; however, it established latency in nearly 90% of ganglia tested. Studies of the phosphorylation of wild-type and d8-10 ICP4 proteins revealed that the serine-rich region is a major determinant for phosphorylation of ICP4 in vivo and that the phosphorylation state could change as a function of the PKA activity. Consistent with this observation, the serine-rich region of ICP4 was shown to be a target for PKA in vitro. While intact ICP4 was readily phosphorylated by ICP4 in vitro, the d8-10 mutant ICP4 was not. Moreover, a synthethic peptide representing a sequence in the serine tract that is predicted to be a substrate for PKA was phosphorylated by PKA in vitro, having a Km within the physiological range. These data suggest that PKA plays a role in viral growth through phosphorylation of one or more sites on the ICP4 molecule.
Multidrug-resistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy
Authors: Iversen A K , Shafer R W , Wehrly K , Winters M A , Mullins J I , Chesebro B , Merigan T C
Published: January 2020

Multidrug-resistant human immunodeficiency virus type 1 (HIV-1) strains with reverse transcriptase (RT) mutations at codons A62-->V, V75-->I, F77-->L, F116-->Y, and Q151-->M have been reported in patients receiving combination therapy with zidovudine (AZT) and didanosine (ddI). Infectious clones with each mutation alone, all five mutations together, and various combinations of mutations were created by site-directed mutagenesis. Mutation Q151-->M conferred partial resistance to AZT, ddI, zalcitibine, and stavudine, whereas a combination of four mutations conferred increased resistance to AZT, ddI, zalcitibine, and stavudine. The positions of residues 75, 77, and 151 in the three-dimensional crystal structure of HIV-1 RT suggest that these residues may affect the ability of the enzyme to discriminate between deoxynucleoside triphosphates and nucleoside analog RT inhibitors. Replication experiments showed that clones with mutation F77-->L but without V75-->I (HIV-1(77), HIV-1(77,151), and HIV-1(77,116,151) had attenuated growth compared with that of the original HIV-1NL4-3 strain and strains containing mutations at both positions 75 and 77 (HIV-1(75,77,151) and HIV-1(75,77,116,15)). Sequence analysis of viral RNA and proviral DNA from several patients indicated that RT mutations developed in a sequential and cumulative pattern over the course of a 2- to 4-year observation period. The present results suggest that drug resistance and viral replicative capacity both may play a role in selection of HIV-1 RT mutations.
The major transcriptional transactivation domain of simian virus 40 large T antigen associates nonconcurrently with multiple components of the transcriptional preinitiation complex
Authors: Johnston S D , Yu X M , Mertz J E
Published: January 2020

Simian virus 40 (SV40) large T antigen (Tag) is a promiscuous transcriptional transactivator; however, its mechanism of transactivation remains unknown. Recent studies have suggested the possible involvement of protein-protein interactions with TBP, the TATA box-binding protein of TFIID, and TEF-1, an enhancer-binding factor. We show here that (i) the Tag domain containing amino acids 133 to 249 directly interacts with the general transcription factor TFIIB, the activator protein Sp1, and the 140-kDa subunit of RNA polymerase II, as well as with TBP and TEF-1; (ii) these interactions can also occur when these transcription factors are present in their functional states in cellular extracts; (iii) binding of Tag to TBP is eliminated by preincubation of TBP either at 48 degrees C or with the adenovirus 13S E1a protein; (iv) this domain of Tag cannot bind concurrently to more than one of these transcription factors; and (v) the substitution of Tag amino acid residues 173 and 174 inactivates the ability of this Tag domain both to associate with any of these transcription factors and to transactivate the SV40 late promoter. Thus, we conclude that SV40 Tag probably does not transactivate via the concurrent interaction with multiple components of the preinitiation complex. Rather, we hypothesize that transactivation by Tag may primarily occur by removing or preventing the binding of factors that inhibit the formation of preinitiation complexes.
Major histocompatibility complex class II I-E-independent transmission of C3H mouse mammary tumor virus
Authors: Wrona T , Dudley J P
Published: January 2020

C57BL/6 mice are resistant to C3H mouse mammary tumor virus (MMTV)-induced mammary tumorigenesis and lack major histocompatibility complex class II I-E molecules that are essential for presentation of C3H superantigen to T cells. T cells are needed for transmission of milk-borne MMTV from the gut to the mammary gland. In this report, we show that infectious C3H MMTV is produced by C57BL/6 mice that nurse on C3H mothers but that virus production in the mammary gland is delayed compared with that in I-E+ mouse strains.
Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection
Authors: Leavitt A D , Robles G , Alesandro N , Varmus H E
Published: January 2020

The viral integrase (IN) protein is the only viral protein known to be required for integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host cell DNA, a step in the viral life cycle that is essential for viral replication. To better understand the relationship between in vitro IN activity and IN-mediated integration of viral DNA in an infected cell, we characterized the effects of 13 IN mutations on viral replication in cultured cells. Using HIV-1 genomes that express the hygromycin resistance gene and do not express the HIV-1 env gene, we generated stocks of pseudotype virus coated with the murine leukemia virus amphotropic envelope glycoprotein, containing either wild-type or mutant HIV-1 IN. All mutants produced normal amounts of physical particles, as measured by reverse transcriptase activity and capsid protein (p24) concentration, but they formed three groups based on infectious titer and synthesis of viral DNA. Changes at the three highly conserved acidic residues in the IN core domain (D-64, D-116, and E-152) impair provirus formation without affecting viral DNA synthesis or the accumulation of viral DNA in the nucleus of the infected cell, a phenotype predicted by each mutant's lack of in vitro integrase activity. Mutations at positions N-120, R-199, and W-235 minimally affect in vitro integrase activity, but infectious titers are severely reduced, despite normal synthesis of viral DNA, implying a defect during integration in vivo. Mutations in the zinc binding region (H12C, H16V, and H16C), S81R, and a deletion of residues 32 through 275 yield noninfectious particles that synthesize little or no viral DNA following infection, despite wild-type levels of reverse transcriptase activity and viral RNA in the particles. The two latter classes of mutants suggest that IN can affect DNA synthesis or integration during infection in ways that are not appreciated from currently used assays in vitro.
The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication
Authors: Mitnaul L J , Castrucci M R , Murti K G , Kawaoka Y
Published: January 2020

In this study, we investigated the role of the conserved neuraminidase (NA) cytoplasmic tail residues in influenza virus replication. Mutants of influenza A virus (A/WSN/33 [H1N1]) with deletions of the NA cytoplasmic tail region were generated by reverse genetics. The resulting viruses, designated NOTAIL, contain only the initiating methionine of the conserved six amino-terminal residues. The mutant viruses grew much less readily and produced smaller plaques than did the wild-type virus. Despite similar levels of NA cell surface expression by the NOTAIL mutants and wild-type virus, incorporation of mutant NA molecules into virions was decreased by 86%. This reduction resulted in less NA activity per virion, leading to the formation of large aggregates of progeny mutant virions on the surface of infected cells. A NOTAIL virus containing an additional mutation (Ser-12 to Pro) in the transmembrane domain incorporated three times more NA molecules into virions than did the NOTAIL parent but approximately half of the amount incorporated by the wild-type virus. However, aggregation of the progeny virions still occurred at the cell surface. All NOTAIL viruses were attenuated in mice. We conclude that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus. In addition, the relative abundance of long filamentous particles formed by the NOTAIL mutants, compared with the largely spherical wild-type particles, indicates a role for the NA cytoplasmic tail in virion morphogenesis.
An array of murine leukemia virus-related elements is transmitted and expressed in a primate recipient of retroviral gene transfer
Authors: Purcell D F , Broscius C M , Vanin E F , Buckler C E , Nienhuis A W , Martin M A
Published: January 2020

Direct RNA-PCR analyses of T-cell lymphomas that developed in rhesus macaques during a gene transfer experiment revealed the presence of several different recombinant murine leukemia viruses (MuLV). Most prominent was the expected MuLV recombinant, designated MoLTRAmphoenv in which the amphotropic env of the helper packaging virus was joined to the long terminal repeat (LTR) of the Moloney MuLV-derived vector. This retrovirus does not exist in nature. An additional copy of the core enhancer acquired from the vector LTR may have augmented the replicative properties of MoLTRAmphoenv MuLV in several different rhesus cell types compared with the prototype amphotropic MuLV4070A. Unexpectedly, at least two types of mink cell focus-forming MuLV elements, arising from endogenous retroviral sequences expressed in the murine packaging cell line, were also transmitted and highly expressed in one of the macaques. Furthermore, murine virus-like VL-30 sequences were detected in the rhesus lymphomas, but these were not transcribed into RNA. The unanticipated presence of an array of MuLV-related structures in a primate gene transfer recipient demands ever-vigilant scrutiny for the existence of transmissible retroviral elements and replication-competent viruses possessing altered tropic or growth properties in packaging cells producing retroviral vectors.
PCR-in situ hybridization detection of human T-cell lymphotropic virus type 1 (HTLV-1) tax proviral DNA in peripheral blood lymphocytes of patients with HTLV-1-associated neurologic disease
Authors: Levin M C , Fox R J , Lehky T , Walter M , Fox C H , Flerlage N , Bamford R , Jacobson S
Published: January 2020

PCR-in situ hybridization (PCR-ISH) was developed and utilized to determine the distribution of human T-cell lymphotropic virus type 1 (HTLV-1) tax proviral DNA in peripheral blood lymphocytes (PBL) from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). PCR-ISH of HTLV-1 tax DNA in PBL from patients with HAM/TSP revealed that 1 in 5,000 to 1 in 10,000 PBL contained virus. PCR-ISH was sensitive, because a positive signal was consistently demonstrated from the HTLV-1-infected cell lines HUT-102 (which contains four to six copies of HTLV-1 proviral DNA per cell) and MT-1 (which contains one to three copies of HTLV-1 proviral DNA per cell). Also, intracellular amplification by PCR-ISH significantly increased sensitivity compared with conventional ISH and was shown to be specific for HTLV-1 tax DNA. These results are in contrast to solution-phase PCR amplification in which greater than 1% of cells were estimated to be infected. The discordance between these results is discussed and may indicate that more than one copy of HTLV-1 tax proviral DNA is present in an individual PBL.
Immunization with viruslike particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus
Authors: Christensen N D , Reed C A , Cladel N M , Han R , Kreider J W
Published: January 2020

Rabbits were immunized with recombinant baculovirus-produced virus-like particles (VLPs) of cottontail rabbit papillomavirus (CRPV) to determine whether these antigens could induce long-term protection against experimental challenge with CRPV. Infectious CRPV and human papillomavirus type 11 L1 VLPs were used as positive and negative control immunogens, respectively. Three groups of immunized animals were challenged with 10-fold serial dilutions of infectious CRPV at 2 weeks, 6 months, and 12 months after immunizations. Antibody titers in serum reached 1:10,000 immediately after the final booster immunization and then decayed to 1:150 at 6 months and 1:100 at 12 months in unchallenged rabbits. Serum neutralization titers followed similar kinetics. Papillomas grew on control-immunized rabbits at sites challenged with 10(-1) (100% of sites), 10(-2) (96% of sites), 10(-3) (63% of sites), and 10(-4) (13% of sites) dilutions of virus. At 2 weeks after CRPV L1 VLP immunizations, the rabbits were completely protected against virus challenge. At both 6 and 12 months after CRPV L1 VLP immunizations, strong protection was also observed. In the last two groups, three of seven rabbits were completely protected and only 4 of 14 or 29% of sites challenged with 10(-1 dilution of virus grew papillomas. Papillomas growing at these four sites were also reduced in size (3.5 +/- 0.7 mm) at 50 days postchallenge compared with sites challenged with 10(-1) dilution on control-immunized rabbits (13.2 +/- 4.2 mm). The results demonstrate that strong and long-lasting protection against experimental challenge with papillomaviruses can be achieved with VLP immunogens.
Recovery and characterization of a replicase complex in rotavirus-infected cells by using a monoclonal antibody against NSP2
Authors: Aponte C , Poncet D , Cohen J
Published: January 2020

Replication of the rotavirus genome involves two steps: (i) transcription and extrusion of transcripts and (ii) minus-strand RNA synthesis in viral complexes containing plus-strand RNA. In this study, we showed evidence for the importance of the viral nonstructural protein of rotavirus, NSP2, in the replication of viral RNAs. RNA-binding properties of NSP2 were tested by UV cross-linking in vivo (in rotavirus-infected MA104 cells and recombinant baculovirus-expressing NSP2-infected Sf9 cells). In rotavirus-infected cells, NSP2 is bound to the 11 double-stranded RNA genomic segments of rotavirus. Quantitative analysis (using hydrolysis by RNase A) is consistent with NSP2 being directly bound to partially replicated viral RNA. Using various monoclonal antibodies and specific antisera against the structural (VP1, VP2, and VP6) and nonstructural (NSP1, NSP2, NSP3, and NSP5) proteins, we developed a solid-phase assay for the viral replicase. In this test, we recovered a viral RNA-protein complex with replicase activity only with a monoclonal antibody directed against NSP2. Our results indicated that these viral complexes contain the structural proteins VP1, VP2, and VP6 and the nonstructural protein NSP2. Our results show that NSP2 is closely associated in vivo with the viral replicase.
Identification of an essential region for internal initiation of translation in the aphthovirus internal ribosome entry site and implications for viral evolution
Authors: Martínez-Salas E , Regalado M P , Domingo E
Published: January 2020

Translation of aphthovirus RNA is initiated at an internal ribosome entry site (IRES) element, preceding the first functional AUG initiation codon. The effect of mutations at the base of domain 3 of the aphthovirus IRES on translation activity has been analyzed by site-directed mutagenesis and expression of bicistronic RNAs in transfected cells. The results have shown that the enhanced IRES activity associated with a single pyrimidine transition fixed in a persistent aphthovirus variant (E. Martínez-Salas, J. C. Sáiz, M. Dávila, G. J. Belsham, and E. Domingo, J. Virol. 67:3748-3755, 1993) is base specific. Mutations predicted to destabilize the base of domain 3 were detrimental to IRES function, but subsequent restoration of the RNA structure gave rise to fully competent IRES. In contrast, single or multiple mutations that did not affect predicted helical structures modified the relative efficiency of translation by at most 10-fold, suggesting that primary sequence also plays a role in IRES activity. A correlation between the energy of stabilization of the IRES structure and the efficiency of translation has been noted. None of the 15 mutations studied reached a level of initiation of translation comparable to that of the IRES from the persistent variant. The results indicate a critical participation of the base of domain 3 in the activity of the aphthovirus IRES, with a strong effect of secondary or higher-order structures and minor effects of primary structure.
Murine cytomegalovirus with a deletion of genes spanning HindIII-J and -I displays altered cell and tissue tropism
Authors: Cavanaugh V J , Stenberg R M , Staley T L , Virgin H W , MacDonald M R , Paetzold S , Farrell H E , Rawlinson W D , Campbell A E
Published: January 2020

Murine cytomegalovirus (MCMV) gene products dispensable for growth in cell culture are likely to have important functions within the infected host, influencing tissue tropism, dissemination, or immunological responses against the virus. To identify such genes, our strategy was to delete large regions of the MCMV genome likely to contain genes nonessential for virus replication in NIH 3T3 cells. Mutant virus RV7 contained a deletion of 7.7 kb spanning portions of MCMV HindIII-J and -I. This virus grew comparably to wild-type (WT) virus in NIH 3T3 fibroblasts, primary embryo fibroblasts, and bone marrow macrophages. However, RV7 failed to replicate in target organs of immunocompetent BALB/c mice and severe combined immunodeficient mice, which are exquisitely sensitive to MCMV infection. This defect in vivo growth may be related to the observation that RV7 grew poorly in the peritoneal macrophage cell line IC-21, which is highly permissive for growth of WT MCMV. Two other mutant viruses with an insertion or smaller deletion in the region common to the RV7 deletion grew comparably to WT virus in the macrophage cell line and replicated in salivary gland tissue. The poor growth of RV7 in IC-21 cells was due to a block in immediate-early gene expression, as levels of RNA from immediate-early gene IE1 were reduced eightfold compared with levels for WT virus in macrophages infected with RV7. Consequently, levels of RNA from early and late genes were also reduced. The lower expression of IE1 in RV7-infected IC-21 macrophages was not due to defective entry of virus into the cells, as equal amounts of viral DNA were present in cells 3 h after infection with RV7 or WT MCMV. These studies demonstrate that deletion of sequences in HindIII-J and -I confer altered cell and tissue tropism.
Apoptosis is induced by N-myc expression in hepatocytes, a frequent event in hepadnavirus oncogenesis, and is blocked by insulin-like growth factor II
Authors: Ueda K , Ganem D
Published: January 2020

Induction of hepatocellular carcinoma in woodchucks by woodchuck hepatitis virus is associated with the activation of N-myc gene expression, usually by viral DNA integration in cis to the N-myc locus. We have examined the consequences of N-myc up-regulation in rodent hepatic cells in culture. Mouse alpha ML hepatocytes infected with a retroviral vector overexpressing the woodchuck N-myc2 gene display a higher proliferation rate than parental alpha ML cells but are morphologically unchanged and do not form colonies in soft agar. However, they display an increased propensity to undergo apoptosis, an effect that is markedly augmented by serum deprivation. Expression of the woodchuck hepatitis virus X gene in alpha ML cells does not alter the growth phenotype of the cells and has no effect upon N-myc-dependent apoptosis. However, apoptosis in N-myc2-expressing alpha ML cells is strongly inhibited by insulin-like growth factor II (IGF II). IGF II gene expression is also strongly up-regulated during hepatic carcinogenesis in vivo in virally infected animals and has been speculated to be part of an autocrine growth-stimulatory pathway. Our results suggest that IGF II may play another role in the development of virus-induced hepatoma: the prevention of programmed cell death triggered by deregulated N-myc expression.
A preferred region for recombinational patch repair in the 5' untranslated region of primer binding site-impaired murine leukemia virus vectors
Authors: Mikkelsen J G , Lund A H , Kristensen K D , Duch M , Sørensen M S , Jørgensen P , Pedersen F S
Published: January 2020

Transduction of primer binding site-impaired Akv murine leukemia virus-based retroviral vectors from the murine packaging cell lines psi-2 and omega E was studied. The efficiency of transduction of the neo marker of all mutated constructs was found to decrease by 5 to 6 orders of magnitude compared with that of the wild-type vector. Thirty-two of 60 transduced proviruses analyzed harbored a primer binding site sequence matching a glutamine tRNA primer. Sequence analysis of the regions flanking the glutamine tRNA primer binding site revealed a distinct pattern of nucleotide differences from the Akv-based vector, suggesting the involvement of a specific endogenous virus-like sequence in patch repair rescue of the primer binding site mutants. The putative recombination partner RNA was found in virions from psi-2 cells as detected by analysis of glutamine tRNA-initiated cDNA and by sequence analysis of regions at or around the glutamine tRNA primer binding site. We propose that the forced recombination of primer binding site mutants involves initial priming on endogenous viral sequences and requires template switching during minus-strand synthesis in the region between the neo gene and the mutated primer binding site to allow correct second-strand transfer in reverse transcription. The system thereby selects for a reverse transcriptase-mediated recombination event in the 5' untranslated region. A panel of sequence differences between the recombination partners in this region has allowed mapping of the site of recombination for each transduction event. Interestingly, the majority of the recombination events were clustered within a narrow, 33-nucleotide region though to be involved in genomic RNA dimerization.
Modulation by gamma interferon of antiviral cell-mediated immune responses in vivo
Authors: Utermöhlen O , Dangel A , Tárnok A , Lehmann-Grube F
Published: January 2020

Mice were infected with lymphocytic choriomeningitis virus and injected once 24 h later with a monoclonal antibody directed against gamma interferon. In comparison with controls, the increase of numbers of CD8+ T cells and the generation of virus-specific cytotoxic T lymphocytes in spleens and virus clearance from organs were diminished, as was the ability of spleen cells to transmit adoptive immunity to infected recipients. The same treatment slightly but consistently lessened rather than augmented the virus titers early in infection, which was also observed in thymusless nu/nu mice. Injection into infected mice of the lymphokine itself in quantities probably higher than are produced endogenously resulted in lower virus titers in spleens but higher titers in livers. The adoptive immunity in infected mice achieved by infusion of immune spleen cells was not altered by treating the recipients with gamma interferon monoclonal antibody. Such treatment did not measurably affect the production of antiviral serum antibodies. We conclude that in lymphocytic choriomeningitis virus-infected mice, gamma interferon is needed for the generation of antivirally active CD8+ T lymphocytes, and furthermore that in this experimental model, direct antiviral effects of the lymphokine elude detection.
Sequences within a small yeast RNA required for inhibition of internal initiation of translation: interaction with La and other cellular proteins influences its inhibitory activity
Authors: Das S , Kenan D J , Bocskai D , Keene J D , Dasgupta A
Published: January 2020

We recently reported purification, determination of the nucleotide sequence, and cloning of a 60-nucleotide RNA (I-RNA) from the yeast Saccharomyces cerevisiae which preferentially blocked cap-independent, internal ribosome entry site (IRES)-mediated translation programmed by the poliovirus (PV) 5' untranslated region (UTR). The I-RNA appeared to inhibit IRES-mediated translation by virtue of its ability to bind a 52-kDa polypeptide which interacts with the 5' UTR of viral RNA. We demonstrate here that the HeLa 52-kDa I-RNA-binding protein is immunologically identical to human La autoantigen. Moreover, I-RNA-mediated purified La protein. By using I-RNAs with defined deletions, we have identified sequences of I-RNA required for inhibition of internal initiation of translation. Two smaller fragments of I-RNA (16 and 25 nucleotides) inhibited PV UTR-mediated translation from both monocistronic and bicistronic RNAs. When transfected into HeLa cells, these derivatives of I-RNA inhibited translation of PV RNA. A comparison of protein binding by active and inactive I-RNA mutants demonstrates that in addition to the La protein, three other polypeptides with apparent molecular masses of 80, 70, and 37 kDa may influence the translation-inhibitory activity of I-RNA.
Molecular genetic analysis of Epstein-Barr virus Cp promoter function
Authors: Evans T J , Farrell P J , Swaminathan S
Published: January 2020

The Cp promoter of Epstein-Barr virus (EBV) directs most transcription of the EBNA genes in lymphoblastoid cell lines. The functions of two control regions in the Cp promoter have been studied by construction of recombinant EBV strains containing specific mutations in these elements. Mutation of the RBP-Jk (CBF1) binding site reduced but did not completely abolish EBNA-2-dependent Cp activity in transient transfection assays. The same mutation in recombinant virus gave only a modest average reduction in Cp function, ranging from full activity to almost no activity in different isolates. Separate deletion of a 262-bp region containing glucocorticoid response elements had little effect in a transient assay but caused a fivefold increase in the steady-state level of Cp RNA in recombinant virus. The results indicate that other elements in addition to the intensively studied RBP-Jk site are important in determining Cp activity in the whole virus. Clonal EBV-infected cell lines expressed RNA from both the Cp and Wp promoters, but the level of Wp RNA did not simply compensate for changes in the level of Cp RNA. The levels of EBNA proteins varied much less than the levels of Cp and Wp RNA, suggesting other types of control in addition to initiation of transcription. A survey of RNAs derived from the internal repeat region of the virus indicated that gene expression from this region of EBV in lymphoblastoid cell lines is accounted for by the known transcripts.
Disruption of prion rods generates 10-nm spherical particles having high alpha-helical content and lacking scrapie infectivity
Authors: Riesner D , Kellings K , Post K , Wille H , Serban H , Groth D , Baldwin M A , Prusiner S B
Published: January 2020

An abnormal isoform of the prion protein (PrP) designated PrPSc is the major, or possibly the only, component of infectious prions. Structural studies of PrPSc have been impeded by its lack of solubility under conditions in which infectivity is retained. Among the many detergents examined, only treatment with the ionic detergent sodium dodecyl sulfate (SDS) or Sarkosyl followed by sonication dispersed prion rods which are composed of PrP 27-30, an N-terminally truncated form of PrPSc. After ultracentrifugation at 100,000 x g for 1 h, approximately 30% of the PrP 27-30 and scrapie infectivity were found in the supernatant, which was fractionated by sedimentation through 5 to 20% sucrose gradients. Near the top of the gradient, spherical particles with an observed sedimentation coefficient of approximately 6S, approximately 10 mm in diameter and composed of four to six PrP 27-30 molecules, were found. The spheres could be digested with proteinase K and exhibited little, if any, scrapie infectivity. When the prion rods were disrupted in SDS and the entire sample was fractionated by sucrose gradient centrifugation, a lipid-rich fraction at the meniscus composed of fragments of rods and heterogeneous particles containing high levels of prion infectivity was found. Fractions adjacent to the meniscus also contained spherical particles. Circular dichroism of the spheres revealed 60% alpha-helical content; addition of 25% acetonitrile induced aggregates high in beta sheet but remaining devoid of infectivity. Although the highly purified spherical oligomers of PrP 27-30 lack infectivity, they may provide an excellent substrate for determining conditions of renaturation under which prion particles regain infectivity.
Theiler's virus persistence and demyelination in major histocompatibility complex class II-deficient mice
Authors: Njenga M K , Pavelko K D , Baisch J , Lin X , David C , Leibowitz J , Rodriguez M
Published: January 2020

Mice with targeted disruption of the A beta gene of major histocompatibility complex class II molecules (Abo) were used to investigate the role of class II gene products in resistance or susceptibility to virus-induced chronic demyelination in the central nervous system (CNS). Class-II-deficient mice from the resistant H-2b [H-2b(Abo)] and nonmutant H-2b backgrounds were infected with Theiler's murine encephalomyelitis virus intracerebrally and examined for CNS virus persistence, demyelination, and neurologic clinical signs. Virus titers measured by plaque assays showed that 8 of 10 normally resistant nonmutant H-2b mice had cleared the virus within 21 days, whereas the other 2 mice had low titers. In contrast, all class II-deficient Abo mice had high virus titers for up to 90 days after infection (4.30 log10 PFU per g of CNS tissue). Virus antigens and RNA were localized to the brains (cortex, hippocampus, thalamus, and brain stem) and spinal cords of Abo mice. Colocalization identified persistent Theiler's murine encephalomyelitis virus in oligodendrocytes and astrocytes but not in macrophages. There was demyelination in 11 of 23 and 6 of 9 Abo mice 45 and 90 days after virus infection, respectively, whereas no demyelination was observed in infected nonmutant H-2b mice. Demyelinating lesions in Abo mice showed virus-specific CD8+ T cells and macrophages but no CD4+ T cells. Spasticity and paralysis were observed in chronically infected Abo mice but not in the nonmutant H-2b mice. These findings demonstrate that class II gene products are required for virus clearance from the CNS but not for demyelination and neurologic disease.
Entry kinetics and mouse virulence of Ross River virus mutants altered in neutralization epitopes
Authors: Vrati S , Kerr P J , Weir R C , Dalgarno L
Published: January 2020

Previously we identified the locations of three neutralization epitopes (a, b1 and b2) of Ross River virus (RRV) by sequencing a number of variants resistant to monoclonal antibody neutralization which were found to have single amino acid substitutions in the E2 protein (S. Vrati, C.A. Fernon, L. Dalgarno, and R.C. Weir, Virology 162:346-353, 1988). We have now studied the biological properties of these variants in BHK cells and their virulence in mice. While variants altered in epitopes a and/or b1 showed no difference, variants altered in epitope b2, including a triple variant altered in epitopes a, b1, and b2, showed rapid penetration but retarded kinetics of growth and RNA and protein synthesis in BHK cells compared with RRV T48, the parent virus. Variants altered in epitopes a and/or b1 showed no change in mouse virulence. However, two of the six epitope b2 variants examined had attenuated mouse virulence. They had a four- to fivefold-higher 50% lethal dose (LD50), although no change in the average survival time of infected mice was observed. These variants grew to titers in mouse tissues similar to those of RRV T48. The ID50 of the triple variant was unchanged, but infected mice had an increased average survival time. This variant produced lower levels of viremia in infected mice. On the basis of these findings we propose that both the receptor binding site and neutralization epitopes of RRV are nearby or in the same domain of the E2 protein.