bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription of a vaccinia virus late promoter template: requirement for the product of the A2L intermediate-stage gene
Authors: Passarelli A L , Kovacs G R , Moss B
Published: January 2020

Evidence is presented that a 26-kDa protein encoded by the vaccinia virus A2L open reading frame, originally shown to be one of three intermediate-stage genes that together can transactivate late-stage gene expression in transfection assays (J. G. Keck, C. J. Baldick, and B. Moss, Cell 61:801-809, 1990), is required for in vitro transcription of a template with a late promoter. The critical step in this analysis was the preparation of an extract containing all the required factors except for the A2L protein. This extract was prepared from cells infected with a recombinant vaccinia virus expressing the bacteriophage T7 RNA polymerase in the presence of the DNA synthesis inhibitor cytosine arabinoside and transfected with plasmids containing the two other known transactivator genes, A1L and G8R, under T7 promoter control. Reaction mixtures made with extracts of these cells had background levels of late transcription activity, unless they were supplemented with extracts of cells transfected with the A2L gene. Active transcription mixtures were also made by mixing extracts from three sets of cells, each transfected with a gene (A1L, A2L, or G8R) encoding a separate factor, indicating the absence of any requirement for their coexpression. To minimize the possibility that the A2L protein functions indirectly by activating another viral or cellular protein, this gene was expressed in insect cells by using a baculovirus vector. The partially purified recombinant protein complemented the activity of A2L-deficient cell extracts. Recombinant A1L, A2L, and G8R proteins, all produced in insect cells, together complemented extracts from mammalian cells containing only viral early proteins, concordant with previous in vivo transfection data.
cis-acting elements in human immunodeficiency virus type 1 RNAs direct viral transcripts to distinct intranuclear locations
Authors: Berthold E , Maldarelli F
Published: January 2020

Two distinct intranuclear locations were identified for alternatively spliced RNA transcripts expressed from the pNL4-3 infectious molecular clone of human immunodeficiency virus (HIV) type 1. Multiply spliced HIV RNA encoding tat was detected within the nucleus in large clusters; immunostaining and colocalization studies using laser-scanning confocal microscopy revealed that these structures contained the non-small nuclear ribonucleoprotein RNA processing factor, SC35. In contrast, unspliced gag RNA was detected in much smaller granules distributed throughout the nucleus, with little or no association with SC35-containing granules. Analyses of nuclear RNA expressed from recombinant plasmids encoding gag (pCMVgag-2) alone or tat (pCMVtat-2) alone revealed distributions corresponding to those obtained with pNL4-3, indicating that expression within the context of the HIV provirus was not required for the distinct RNA locations detected for these transcripts. The presence of unspliced gag RNA in small granules was confirmed in infections of H9 T-lymphocytic cells, indicating that gag localization was not restricted to transient expression systems. The intranuclear distribution of gag RNA was dependent on specific RNA sequences. Deletion of a portion of the gag gene of pCMVgag-2, containing a cis-repressing inhibitory region, resulted in redirection of unspliced gag RNA from small granules into large SC35-containing clusters. The addition of the Rev-responsive element, RRE, to the deleted pCMVgag-2 construct resulted in RNA transcripts which were no longer associated with SC35. We also identified a cellular intron, rabbit beta-globin-intervening sequence 2 (IVS-2) which, when introduced into pCMVgag-2, redirected unspliced gag RNA into SC35-containing granules and permitted rev-independent Gag expression. These findings suggest that redirecting intranuclear RNA localization may influence gene expression. Color micrographs from this article are available for view at http//128.231.216.2/lmmhome.htm.
The polymerase-like core of brome mosaic virus 2a protein, lacking a region interacting with viral 1a protein in vitro, maintains activity and 1a selectivity in RNA replication
Authors: Smirnyagina E , Lin N S , Ahlquist P
Published: January 2020

Brome mosaic virus (BMV), a member of the alphavirus-like super-family of positive-strand RNA viruses, encodes two proteins required for viral RNA replication: 1a and 2a. 1a contains m7G methyltransferase- and helicase-like domains, while 2a contains a polymerase (pol)-like core flanked by N- and C-terminal extensions. Genetic studies show that BMV RNA replication requires 1a-2a compatibility implying direct or indirect 1a-2a interaction in vivo. In vitro, la interacts with the N-terminal 125-amino-acid segment of 2a preceding the pol-like core, and prior deletion studies suggested that this 2a segment was essential for RNA replication. We have now used protein fusions and deletions to explore possible parallels between noncovalent 1a-2a interaction and covalent fusion of similar protein domains in tobacco mosaic virus and to see whether the N-terminal 2a-1a interaction was the primary basis for 1a-2a compatibility in vivo. We found that 2a can function as part of a tobacco mosaic virus-like 1a-2a fusion and that a 2a segment (amino acids 162 to 697) comprising the pol-like core was sufficient to provide 2a functions in such a fusion. Unexpectedly, the unfused 2a core segment also supported RNA replication when it and wild-type la were expressed as separate proteins. Moreover, in gene reassortant experiments with the related cowpea chlorotic mottle virus, the unfused 2a core segment showed the same 1a compatibility requirements as did wild-type BMV 2a. Thus, the pol-like core of 2a must interact with la in a way that is selective and essential for RNA synthesis, and 1a-2a interactions are more complex than the single, previously mapped interaction of the N-terminal 2a segment with 1a.
Evidence that the nuclease activities associated with the herpes simplex type 1 DNA polymerase are due to the 3'-5' exonuclease
Authors: Hall J D , Orth K L , Claus-Walker D
Published: January 2020

We investigated nuclease activities associated with the catalytic subunit of herpes simplex virus type 1 DNA polymerase. We confirm that a 3'-5' exonuclease copurifies with this enzyme. Previous reports suggested that a 5' DNase was intrinsic to the polymerase. Our preparation lacks such activity.
The concentration of Ca2+ that solubilizes outer capsid proteins from rotavirus particles is dependent on the strain
Authors: Ruiz M C , Charpilienne A , Liprandi F , Gajardo R , Michelangeli F , Cohen J
Published: January 2020

It has been previously shown that rotavirus maturation and stability of the outer capsid are calcium-dependent processes. More recently, it has been hypothesized that penetration of the cell membrane is also affected by conformational changes of the capsid induced by Ca2+. In this study, we determined quantitatively the critical concentration of calcium ion that leads to solubilization of the outer capsid proteins VP4 and VP7. Since this critical concentration is below or close to trace levels of Ca2+, we have used buffered solutions based on ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca-EGTA. This method allowed us to show a very high variability of the free [Ca2+] needed to stabilize, at room temperature, the outer capsid of several rotavirus strains. This concentration is about 600 nM for the two bovine strains tested (RF and UK), 100 nM for the porcine strain OSU, and only 10 to 20 nM for the simian strain SA11. Titration of viral infectivity after incubation in buffer of defined [Ca2+] confirmed that the loss of infectivity occurs at different [Ca2+] for these three strains. For the bovine strain, the cleavage of VP4 by trypsin has no significant effect on the [Ca2+] that solubilizes outer shell proteins. The outer layer (VP7) of virus-like particles (VLP) made of recombinant proteins VP2, VP6, and VP7 (VLP2/6/7) was also solubilized by lowering the [Ca2+]. The critical concentration of Ca2+ needed to solubilize VP7 from VLP2/6/7 made of protein from the bovine strain is close to the concentration needed for the corresponding virus. Genetic analysis of this phenotype in a set of reassortant viruses from two parental strains having the phenotypes of strains OSU (porcine) and UK (bovine) confirmed that this property of viral particles is probably associated with the gene coding for VP7. The analysis of VLP by reverse genetics might allow the identification of the region(s) essential for calcium binding.
Frequency of multiple Epstein-Barr virus infections in T-cell-immunocompromised individuals
Authors: Yao Q Y , Tierney R J , Croom-Carter D , Dukers D , Cooper G M , Ellis C J , Rowe M , Rickinson A B
Published: January 2020

The Epstein-Barr virus (EBV) carrier state is characterized by latent infection of the general B-cell pool and by chronic virus replication at oropharyngeal sites. In Caucasian populations, most healthy carriers seem to harbor one dominant transforming virus strain, usually of type I rather than type 2, which persists over time and is detectable both in the blood and in the throat. This finding implies that once the virus carrier state is established, both viral reservoirs are largely if not completely protected from infection with additional strains. However, it is not known which facets of the immune response offer that protection. Here we address this question by a detailed study of EBV carriage in patients T-cell immunocompromised as a result of chronic human immunodeficiency virus (HIV) infection. Resident EBV strains were rescued from blood and from throat washings by using an in vitro transformation assay which aims to minimize bias toward faster-growing transformants; in this way, a mean of 16 independent isolations were made from each of 35 HIV-positive (predominantly male homosexual) patients. These virus isolates were characterized first at the DNA level by PCR amplification across type-specific polymorphisms in the EBNA2 and EBNA3C genes and across the 30-bp deletion and 33-bp repeat loci in the LMP1 gene and then at the protein level by immunoblotting for the strain-specific "EBNAprint" of EBNA1, -2, and -3C molecular weights. By these criteria, 18 of 35 patients harbored only one detectable EBV strain, usually of type 1, as do healthy carriers. However, the other 17 patients showed clear evidence of multiple infection with different EBV strains. In eight cases these strains were of the same type, again usually type 1, and were more often found coresident in throat washings than in the blood. By contrast, a further nine patients gave evidence of coinfection with type 1 and type 2 strains, and in these cases both virus types were detectable in the blood as well as in the throat. Immunological assays on these HIV-positive patients as a group showed a marked impairment of T-cell responses, reflected in reduced levels of EBV-specific cytotoxic T-cell memory, but an elevation of humoral responses, reflected in raised antibody titers to the EBV envelope glycoprotein gp340 and by the maintenance of virus neutralizing antibodies in serum. We infer that selective impairment of the T-cell system predisposes the host to infection with additional exogenously transmitted EBV strains.
Among all human T-cell leukemia virus type 1 proteins, tax, polymerase, and envelope proteins are predicted as preferential targets for the HLA-A2-restricted cytotoxic T-cell response
Authors: Pique C , Connan F , Levilain J P , Choppin J , Dokhélar M C
Published: January 2020

The human T-cell leukemia virus type 1 (HTLV-1) is a human retrovirus associated with two diseases for which no successful treatment is yet available; the development of a vaccine is therefore an important issue. Since HTLV-1 is a persistent virus, an efficient vaccine will probably require a cytotoxic T-lymphocyte (CTL) response in addition to the production of antibodies. To identify potential CTL epitopes, we have selected, within all of the HTLV-1 proteins, nonapeptides containing anchor residues required for association with HLA-A2 molecules (residues at positions 2 and 9), which is the most frequently occurring A allele in all human populations. A set of 111 peptides was synthetized and tested in vitro in two assembly assays using processing-defective T2 cells. Anchor motifs selected were those containing two major anchor residues (L2/M2/12-V9/L9/I9) (one letter amino-acid code) and those including tolerated anchor residues (V2/A2/T2 and/or A9/M9/T9). The analysis of the binding capacity of the peptides confirms the high efficiency of the L2-V9 anchor motif and shows that a systematic research of potential binding peptides should exclude peptides containing known detrimental residues rather than select only peptides with known favored residues. We show that 39 peptides representative of all the HTLV-1 proteins are able to bind to HLA-A2 molecules. Strong binder peptides which are very likely good CTL epitopes were identified in three HTLV-1 proteins, Tax, envelope, and polymerase. Three of the strong binder peptides correspond to previously described HLA-A2-restricted CTL epitopes in the Tax protein, and two others are localized in a domain of the viral envelope recognized by natural neutralizing antibodies. This latter result has important implications for the development of an anti-HTLV-1 vaccine.
The N-glycan of the SCR 2 region is essential for membrane cofactor protein (CD46) to function as a measles virus receptor
Authors: Maisner A , Alvarez J , Liszewski M K , Atkinson D J , Atkinson J P , Herrler G
Published: January 2020

Membrane cofactor protein (MCP) (CD46), a complement-regulatory protein, serves as a cellular receptor for measles virus. Its amino-terminal portion is composed of four short consensus repeats (SCR), three of which (SCR1, SCR2, and SCR4) carry an N-linked oligosaccharide. In order to determine the importance of the three N-glycans for the function of MCP as a measles virus receptor, we established Chinese hamster ovary (CHO) cell lines that stably express mutant MCPs lacking one of the three motifs for N glycosylation (NQ1, NQ2, and NQ4). In an additional mutant (NQ1-2), two glycosylation motifs were altered, allowing the addition of an N-linked oligosaccharide only in SCR4. The abilities of the mutant MCPs to function as measles virus receptors were analyzed with three different assays: (i) binding of measles virus hemagglutinin to MCP immobilized on nitrocellulose; (ii) binding of measles virus to CHO cells expressing wild-type or mutant MCP; and (iii) infection of the transfected CHO cells by measles virus. In all three assays, the abilities of the NQ2 and NQ1-2 mutants to serve as measles virus receptors were drastically impaired. The NQ1 and NQ4 mutants were recognized by measles virus almost as efficiently as the wild-type protein. These results indicate that the N-glycan attached to SCR2 is essential for MCP to serve as a measles virus receptor, while the oligosaccharides attached to SCR1 and SCR4 are of only minor importance.
cis-active structural motifs involved in specific encapsidation of Moloney murine leukemia virus RNA
Authors: Mougel M , Zhang Y , Barklis E
Published: January 2020

We have analyzed the roles of RNA structural motifs located in the 5' part of the Moloney murine leukemia virus (M-MuLV) encapsidation domain (Psi region) with regard to their effects on viral replication. Four putative stem-loop structures between the 5' splice donor site and the gag initiation codon have been examined: stem structure A, corresponding to M-MuLV viral nucleotides 211 to 224; stem-loop B, nucleotides 278 to 303; stem-loop C, nucleotides 310 to 352; and stem-loop D, nucleotides 355 to 374. By measuring infectivities, encapsidation and splicing efficiencies, and endogenous reverse transcription levels of motif A, B, C, and D deletion mutants, we identified mutations which affect replication at the encapsidation step. In particular, deletion of all four motifs in a single mutant eliminated encapsidation of viral RNA, while deletion of individual elements moderately reduced the encapsidation efficiencies. Through analysis of different deletion combinations, we found that deletion of the first two motifs (A plus B) reduced both encapsidation and reverse transcription efficiencies, while deletion of the 3' motifs (C plus D) eliminated encapsidation. Interestingly, the C and D motifs both contain a GACG loop sequence and are highly conserved among murine type C retroviruses. Our results indicate that M-MuLV motifs C and D are necessary for efficient encapsidation, and the presence of at least one of these two stem-loops is crucial to encapsidation and virus replication.
Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein
Authors: Bertagnoli S , Gelfi J , Le Gall G , Boilletot E , Vautherot J F , Rasschaert D , Laurent S , Petit F , Boucraut-Baralon C , Milon A
Published: January 2020

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.
A single gene encoding the fiber is responsible for variations in virulence in the fowl adenoviruses
Authors: Pallister J , Wright P J , Sheppard M
Published: January 2020

Intertypic recombinant fowl adenoviruses (FAVs) were generated to determine regions of the viral genome involved in virulence. Recombinants were produced with two serotype 8 FAVs, mildly virulent CFA 3 and hypervirulent CFA 40. Restriction endonuclease fragments from the genomes of the two FAVs were used to transfect primary chicken kidney cells. Virulence testing of these recombinants located the region responsible for differences in virulence to an 8.4-kb fragment of the genome located between kb 26.6 and 35.0. According to data available for a serotype 10 FAV that had been partially characterized in the laboratory, this segment of the genome contained three genes of known identity (100K, 33K, and pVIII) and a region between kb 31 and 35 with unknown coding potential (although this information subsequently became available for a serotype 1 FAV, CELO). Therefore, the region between kb 30.5 and 34.5 was sequenced. The results revealed that the unknown region encoded a fiber gene on the right strand and several small open reading frames of unknown identity on the left strand. Further recombinant viruses containing defined exchanges within the 4-kb fragment were constructed, and virulence testing of these viruses indicated that the fiber was responsible for differences in virulence for CFA 40 and CFA 3.
The HeLa cell receptor for enterovirus 70 is decay-accelerating factor (CD55)
Authors: Karnauchow T M , Tolson D L , Harrison B A , Altman E , Lublin D M , Dimock K
Published: January 2020

Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [35S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70- to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70 and stably, transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.
Proviral burden and infection kinetics of feline immunodeficiency virus in lymphocyte subsets of blood and lymph node
Authors: Dean G A , Reubel G H , Moore P F , Pedersen N C
Published: January 2020

Feline immunodeficiency virus (FIV) is similar to human immunodeficiency virus type 1 virologically and induces a clinical syndrome in cats comparable to human immunodeficiency virus type 1 syndrome in humans. To determine the lymphoid target cells of FIV, populations of CD4+ lymphocytes, CD8+ lymphocytes, and CD21+ lymphocytes (B cells) were enriched to more than 96.5% purity and then analyzed for FIV provirus by semiquantitative DNA amplification. We found FIV provirus in CD4+, CD8+, and B lymphocytes. In cats infected for <4 months, proviral burden was greatest in CD4+ cells, followed by B cells and then by CD8+ cells. In cats infected for more than 5 years, proviral burden was greatest in B cells, followed by CD4+ cells and then by CD8+ cells. The total proviral burden was > 1 log10 higher in acutely infected cats than in chronically infected cats, primarily because of a higher level of CD4+ infection in the acutely infected cats. A comparison of proviral loads in mesenteric lymph node and peripheral blood mononuclear cells in acutely or chronically infected cats revealed no significant difference. A kinetics study of FIV infection demonstrated that all lymphocyte subpopulations were infected by 4 weeks postinoculation. Virus was isolated from CD4+, CD8+, and B cells in vitro, and reverse transcriptase PCR demonstrated that all subsets contained viral RNA in vivo and therefore are productive reservoirs for FIV.
Interaction between hepatitis C virus core protein and E1 envelope protein
Authors: Lo S Y , Selby M J , Ou J H
Published: January 2020

Hepatitis C virus has three structural genes named C, E1, and E2. The C gene encodes the core (capsid) protein and the E1 and E2 genes encode the envelope proteins. In an immunoprecipitation experiment, the E1 protein was found to be precipitated by an anti-core antibody in the presence but not in the absence of the core protein, indicating that the E1 protein can interact with the core protein. This interaction is independent of whether the E1 and the C genes are linked in cis or separated in different DNA constructs for expression. The interaction between the core and the E1 proteins is confirmed by the observation that a hybrid protein derived from the core protein and the tissue plasminogen activator is localized in the nucleus in the absence of the E1 protein and in the perinuclear region in the presence of the E1 protein. Deletion-mapping studies indicate that the carboxy-terminal sequences of both the core and the E1 proteins are important for their interaction. Since little E1 sequence is exposed on the cytosolic side of the membrane of the endoplasmic reticulum, the interaction between the core and the E1 proteins most likely takes place in the endoplasmic reticulum membrane. The E2 protein could not be coprecipitated with the core protein by the anti-core antibody in a similar assay and likely does not interact with the core protein. The implications of these findings on the morphogenesis of the hepatitis C virus virion are discussed.
Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes
Authors: Hanecak R , Brown-Driver V , Fox M C , Azad R F , Furusako S , Nozaki C , Ford C , Sasmor H , Anderson K P
Published: January 2020

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.
Inhibition of human immunodeficiency virus type 1 production in infected peripheral blood mononuclear cells by human leukocyte antigen class I-specific antibodies: evidence for a novel antiviral mechanism
Authors: Briant L , Benkirane M , Girard M , Hirn M , Iosef C , Devaux C
Published: January 2020

A well-characterized mechanism by which anti-HLA class I monoclonal antibodies (MAb) inhibit human immunodeficiency virus type 1 (HIV-1) propagation in in vitro cell cultures is the neutralization of the virus through interactions with HLA molecules associated with the virion envelope. Yet, the possibility that another mechanism of inhibition might affect a postbinding stage of the virus life cycle has been strongly suggested by our previous investigations. To demonstrate that the interaction of MAb B1-1G6 with the light chain of cell surface-expressed HLA class I molecules inhibits a postbinding step of the HIV-1 life cycle, peripheral blood mononuclear cells (PBMCs) were exposed to viruses grown in HLA class I-negative, CD4-positive cells (these viruses, which did not carry HLA class I molecules, cannot be neutralized by anti-HLA MAb during the first round of infection), and PCR was used at various times postexposure to search for the different forms of HIV-1 DNA and RNA in virus-exposed PBMCs cultured in either the presence or [correction of] absence of MAb B1-1G6. Although viral DNA was found in MAb B1-1G6-treated cells, spliced HIV-1 mRNA could not be detected in those cells. In contrast, HIV-1 gene expression was found in HIV-1-infected PBMCs treated with B9-12-1, another HLA class I-specific MAb which prevents infection of cells by cell-free viruses but which fails to inhibit cell-to-cell transmission of HIV-1. These results highlight a second antiviral mechanism by which anti-HLA MAb inhibit in vitro HIV-1 propagation.
Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate
Authors: Jackson T , Ellard F M , Ghazaleh R A , Brookes S M , Blakemore W E , Corteyn A H , Stuart D I , Newman J W , King A M
Published: January 2020

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.
cis-acting elements involved in transcriptional regulation of the herpes simplex virus type 1 latency-associated promoter 1 (LAP1) in vitro and in vivo
Authors: Soares K , Hwang D Y , Ramakrishnan R , Schmidt M C , Fink D J , Glorioso J C
Published: January 2020

Latency-associated promoter 1 (LAP1) of herpes simplex virus type 1 is required to generate a series of latency-associated transcripts (LATs) in sensory neurons of latently infected animals. Sequence analysis and DNA binding studies have suggested the existence of several cis-acting elements within LAP1 that are potentially important for promoter function, although their role in LAT gene expression during latency is largely unexplored. In this report, we present evidence that the LAP1 TATA box is essential for transcription initiation in vitro. A reduction in LAT synthesis measured by in situ hybridization and reverse transcription-PCR (RT-PCR) of rat brain tissue latently infected with a LAP1 TATA substitution virus demonstrated that this sequence was required for full LAP1 activity in vivo. Analysis of additional site-directed and 5'-deletion mutants of LAP1 by in vitro transcription-primer extension assays showed that upstream elements including the USF and cyclic AMP response element (CRE) site specifically contributed to LAP1 function and that sequences beginning at position -620 relative to the transcription start site were essential for full promoter activity. The combination of deleting USF, CRE, and TATA completely abolished LAT expression in the brain, identifying these as essential elements for the neuron-specific functioning of LAP1 during latency. Mutation of the transcription start site did not abolish transcription, suggesting the absence of an initiator element. However, one of the most exciting findings from this study is that the region downstream of the TATA box appears to contain a true enhancer that is not only essential for transcription, but also functional when positioned 1.6 kb downstream of the start site of transcription. It was concluded that (i) the TATA box was essential for full transcriptional activity from LAP1 both in vitro and in vivo, (ii) the USF element and CRE contribute to LAP1 function during latency in combination with the TATA element, (iii) multiple trans-acting factors besides the USF- and CRE-binding proteins were required for full promoter activity in vitro, and (iv) sequences downstream of the TATA box enhanced promoter activity in vitro.
Identification of domains in canine parvovirus VP2 essential for the assembly of virus-like particles
Authors: Hurtado A , Rueda P , Nowicky J , Sarraseca J , Casal J I
Published: January 2020

Canine parvovirus capsids are composed of 60 copies of VP2 and 6 to 10 copies of VPl. To locate essential sites of interaction between VP2 monomers, we have analyzed the effects of a number of VP2 deletion mutants representing the amino terminus and the four major loops of the surface, using as an assay the formation of virus-like particles (VLPs) expressed by recombinant baculoviruses. For the amino terminus we constructed three mutants with progressively larger deletions, i.e., 9, 14, and 24 amino acids. Deletions of 9 and 14 amino acids did not affect the morphology and assembly capabilities of the mutants. However, the mutant with the 24-amino-acid deletion did not show hemagglutination properties or correct VLP morphology, stressing again the relevance of the RNER domain in canine parvovirus functionality. Three of the four mutants with deletions in the loops failed to make correct VLPs, indicating that these regions are essential for correct capsid assembly and morphology. Only the mutant with the deletion in loop 2 was able to assemble in regular VLPs, suggesting that this loop has little or no effect in capsid morphogenesis. Further research has demonstrated that this region can tolerate the insertion of foreign epitopes that are correctly exposed in the surface of the capsid. This result opens the door to the use of these VLPs for antigen delivery.
Transforming growth factor beta1 induces differentiation in human papillomavirus-positive keratinocytes
Authors: Ozbun M A , Meyers C
Published: January 2020

Human papillomaviruses (HPVs) are implicated in the etiology of anogenital cancers. Expression of the HPV E6 and E7 oncoproteins is believed to contribute to the carcinogenic process. Progressive loss of the ability to differentiate and resistance to the growth-inhibitory effects of endogenous signals also appear important in multistep tumorigenesis. Transforming growth factor beta1 (TGF-beta1) is a potent growth inhibitor for a variety of cultured cells. There have been conflicting reports on the ability of TGF-beta1 to inhibit the growth of HPV-positive keratinocytes in monolayer cultures. We have employed the organotypic (raft) tissue culture system, which more accurately mimics the in vivo cellular environment and architecture. We have investigated the TGF-beta1 response of HPV-positive keratinocytes derived from neoplastic cervical biopsies. Growth of these cell lines as raft tissues showed that many were altered in the ability to stratify and synthesize differentiation-specific proteins. When the organotypic tissues were treated with TGF-beta1, a more complete differentiation of the keratinocytes was induced. Treatment with 12-0-tetradecanoylphorbol-13-acetate gave similar results. TGF-beta1 treatment of HPV-positive raft epithelia led to a dose-dependent increase in E7 RNA expression in contrast to results from previous studies with monolayer cultures. Furthermore, TGF-beta1 interfered with the proliferation of HPV-positive cell lines grown in monolayer cultures. Our results suggest that loss of the ability to express markers of differentiation, a characteristic of malignancy, is a two-step process. The first step is reversible; the second is irreversible.
Alteration of intracellular potassium and sodium concentrations correlates with induction of cytopathic effects by human immunodeficiency virus
Authors: Voss T G , Fermin C D , Levy J A , Vigh S , Choi B , Garry R F
Published: January 2020

Increases in intracellular concentrations of potassium ([K+]i) and sodium ([Na+]i) occur concomitantly with cytopathic effects induced in a CD4+ T-lymphoblastoid cell line acutely infected by human immunodeficiency virus (HIV). This [K+]i increase was greater in cells infected by cytopathic HIV strains than in cells infected by less cytopathic strains. T cells persistently infected by HIV had an increased [K+]i but displayed an [Na+]i similar to that of mock-infected cells. HIV induced increases in [K+]i and [Na+]i after cytopathic infection of human peripheral blood mononuclear cells, but the magnitude of the Na+ changes did not correlate with the extent of the cytopathic effect. Enhanced movement of cations may osmotically drive water entry, resulting in balloon degeneration and lysis of HIV-infected cells. These observations offer potential approaches for antiviral therapies.
A putative receptor for Venezuelan equine encephalitis virus from mosquito cells
Authors: Ludwig G V , Kondig J P , Smith J F
Published: January 2020

We have identified a cellular protein from a continuous mosquito cell line (C6/36) that appears to play a significant role in the attachment of Venezuelan equine encephalitis (VEE) virus to these cells. VEE virus bound to a 32-kDa polypeptide present in the C6/36 plasma membrane fraction, and binding to this polypeptide was dose dependent and saturable and competed with homologous and heterologous alphaviruses. These observations suggest that this polypeptide binds virus via a receptor-ligand interaction. The 32-kDa polypeptide was expressed on the surfaces of C6/36 cells, and monoclonal antibodies directed against either this cell polypeptide or the VEE virus E2 glycoprotein, which is thought to be the viral attachment protein, interfered with virus attachment. Collectively, these data provide evidence suggesting that the 32-kDa polypeptide serves as a receptor for VEE virus infection of cells. We have characterized this cell polypeptide as a laminin-binding protein on the basis of its ability to interact directly with laminin as well as its immunologic cross-reactivity with the high-affinity human laminin receptor.
Paradoxical interactions between human delta hepatitis agent RNA and the cellular protein kinase PKR
Authors: Robertson H D , Manche L , Mathews M B
Published: January 2020

The genome of the human delta hepatitis agent is a circular, highly structured single-stranded RNA lacking regular runs of RNA-RNA duplex longer than 15 bp. We have tested the ability of delta agent RNA to participate in reactions with a protein containing a motif which confers the ability to bind double-stranded RNA (dsRNA). Surprisingly, highly purified delta agent RNA preparations from which all traces of contaminating dsRNA have been removed activate PKR, the dsRNA-dependent protein kinase activity of mammalian cells (also known as DAI, P1-eIF-2, and p68 kinase). This behavior is in marked contrast to the interaction of PKR with a number of other highly structured viral single-stranded RNAs, which inhibit, rather than stimulate, activation of this kinase. PKR activation leads to inhibition of protein synthesis in the rabbit reticulocyte lysate system. Paradoxically, delta RNA failed to elicit the expected PKR-mediated inhibition of cell-free translation. Instead, delta RNA interfered with PKR activation and the translational block induced by dsRNA. We conclude that the interaction of PKR and delta agent RNA may represent a new category of protein-RNA interactions involving the dsRNA binding motif.
Analysis of conserved domains of UL41 of herpes simplex virus type 1 in virion host shutoff and pathogenesis
Authors: Strelow L I , Leib D A
Published: January 2020

The herpes simplex virus type 1 virion host shutoff protein has four domains whose sequences are conserved only among neurotropic herpesviruses. Mutant viruses with 29- and 31-amino-acid deletions in domains III and IV but outside of the domain required for interaction with VP16 were generated. The mutants failed to induce cellular RNA degradation and showed impaired virulence in mice. Domains III and IV are therefore required for both shutoff and virulence.
Characterization of a myxoma virus-encoded serpin-like protein with activity against interleukin-1 beta-converting enzyme
Authors: Petit F , Bertagnoli S , Gelfi J , Fassy F , Boucraut-Baralon C , Milon A
Published: January 2020

A genomic library of myxoma virus (MV) DNA, a leporipoxvirus that causes myxomatosis, was constructed and screened by in vitro transcription-translation. A clone was selected on the basis of its strong reactivity with MV antiserum. Analysis of the corresponding DNA sequence and the deduced amino acid sequence revealed an open reading frame coding for a 34-kDa protein with strong homologies to members of the serpin superfamily. The gene encoding this new protein, called serp2, was localized on the MV genome. Interestingly, this gene is deleted in an attenuated strain. We constructed a baculovirus vector to produce recombinant Serp2 protein and raised specific antisera that allowed the characterization of Serp2 expression during the MV cycle. The biological relevance of this new serpin from MV was monitored, and it was shown that Serp2 could inhibit human interleukin-1 beta-converting enzyme activity.
Epstein-Barr virus nuclear protein 2 (EBNA2) binds to a component of the human SNF-SWI complex, hSNF5/Ini1
Authors: Wu D Y , Kalpana G V , Goff S P , Schubach W H
Published: January 2020

Epstein-Barr nuclear antigen 2 (EBNA2), one of the six viral nuclear proteins expressed in latently infected B lymphocytes, is essential to the immortalization of B cells by Epstein-Barr virus (EBV). EBNA2 promotes transcriptional transactivation of viral and cellular genes by acting as an adapter molecule that binds to cellular sequence-specific DNA-binding proteins, JK recombination signal-binding protein (RBP-JK), and PU.1 and engages multiple members of the RNA polymerase II transcription complex. In the present study, we show that EBNA2 also interacts with hSNF5/Ini1, the human homolog of the yeast transcription factor SNF5. Gel filtration fractionation of partially purified EBV-positive lymphocyte nuclear extracts shows that a fraction of EBNA2 coelutes with both hSNF5/Ini1 and BRG1, a human homolog of SWI/SNF2, in the high-molecular-mass region (1.5 to 2.0 MDa) of a Superose 6 chromatogram. An affinity-purified rabbit antibody directed against hSNF5/Ini1 coimmunoprecipitates EBNA2 from this high-molecular-mass nuclear protein fraction, demonstrating that EBNA2 and hSNF5/Ini1 interact in vivo. This interaction is restricted to a subpopulation of phosphorylated viral EBNA2. Deletion mutation analysis of EBNA2 shows that the proline-rich aminoterminal end and a domain within the divergent region of EBNA2 mediate EBNA2-hSNF5/Ini1 interaction. Since the SNF-SWI complex participates in gene regulation through the alteration of nucleosome configuration and may be a component of the RNA polymerase II holoenzyme, the EBNA2-hSNF5/Ini1 interaction supports the hypothesis that EBNA2 facilitates transcriptional transactivation by acting as a transcription adapter molecule. We postulate that EBNA2 engages the hSNF-SWI complex to generate an open chromatin conformation at the EBNA2-responsive target genes, thereby potentiating the function of the RBP-JK-EBNA2-polymerase II transcription complex.
Mutational analysis of the envelope protein of spleen necrosis virus
Authors: Martinez I , Dornburg R
Published: January 2020

Spleen necrosis virus (SNV) is an amphotropic type C retrovirus originally isolated from a duck. The envelope protein is related to that of type D retroviruses, and SNV appears to use the same receptor as do simian retroviruses. However, little is known about envelope-receptor interactions of SNV. We constructed a series of envelope mutants to characterize the SU peptide of SNV. Point mutations were introduced throughout SU in regions that are conserved among all retroviruses belonging to the same receptor interference group. The biological and biochemical properties of these mutants were analyzed. All mutants were transported efficiently to the cell surface. Almost all mutations in the amino-terminal one-third caused a conformational change of the envelope and a significant drop in infectivity and abolished the ability to confer superinfection interference. Similar observations were made with only two of seven mutants with mutations in the middle of SU. Four mutations in this region had little or no effect on biological activity. One mutant envelope protein (Asp to Arg at position 192) was processed normally but showed little infectivity and had no ability to confer superinfection interference. A detailed mutational analysis suggested that this amino acid forms a hydrogen bond to its cellular receptor. Mutations within the carboxy-terminal part of SU had very little or no effect on biological function. Aberrantly processed envelope proteins were proteolytically cleaved at a new point upstream of and differing in sequence from the conserved retroviral SU/TM cleavage site. Surprisingly, these mutants still retained some infectivity (0.01 to 1% of that of the wild type). Our data indicate that the envelope of SNV behaves in a manner very different from that of the envelopes of other studied retroviruses.
Inducible gene expression by retrovirus-mediated transfer of a modified tetracycline-regulated system
Authors: Iida A , Chen S T , Friedmann T , Yee J K
Published: January 2020

The ability to regulate gene expression via exogenous stimuli will facilitate the study of gene functions in mammalian cells. In the present study, we modified the tetracycline-controlled inducible system by the addition of the ligand-binding domain of the estrogen receptor to the carboxy terminus of the tTA transactivator. A single retroviral vector can transduce both the transactivator gene and the gene of interest controlled by the tTA-inducible promoter into mammalian cells. We show that cell lines expressing the transactivator can readily be established and that expression of the gene of interest depends on the removal of tetracycline and the addition of estrogen. By using this system, cell lines with inducible expression of the G protein of vesicular stomatitis virus, a potentially toxic gene product, were established. The combination of a powerful inducible system and retrovirus-mediated gene transfer can not only be used to study gene function but may also be applied in the future to clinical trials in human gene therapy.