bluuebunny/crossref_metadata_2025 huggingface dataset:
A small region of the ecotropic murine leukemia virus (MuLV) gag gene profoundly influences the types of polytropic MuLVs generated in mice
Authors: Lavignon M , Richardson J , Evans L H
Published: January 2020

The vast majority of recombinant polytropic murine leukemia viruses (MuLVs) generated in mice after infection by ecotropic MuLVs can be classified into two major antigenic groups based on their reactivities to two monoclonal antibodies (MAbs) termed Hy 7 and 516. These groups very likely correspond to viruses formed by recombination of the ecotropic MuLV with two distinct sets of polytropic env genes present in the genomes of inbred mouse strains. We have found that nearly all polytropic MuLVs identified in mice infected with a substrain of Friend MuLV (F-MuLV57) are reactive with Hy 7, whereas mice infected with Moloney MuLV (Mo-MuLV) generate major populations of both Hy 7- and 516-reactive polytropic MuLVs. We examined polytropic MuLVs generated in NFS/N mice after inoculation with Mo-MuLV-F-MuLV57 chimeras to determine which regions of the viral genome influence this difference between the two ecotropic MuLVs. These studies identified a region of the MuLV genome which encodes the nucleocapsid protein and a portion of the viral protease as the only region that influenced the difference in polytropic-MuLV generation by Mo-MuLV and F-MuLV57.
Hygromycin B resistance mediates elimination of Leishmania virus from persistently infected parasites
Authors: Ro Y T , Scheffter S M , Patterson J L
Published: January 2020

A series of pX63-HYG derivatives encoding Leishmania RNA virus 1-4 (LRV1-4) sequences were electroporated into cells of Leishmania strain M4147, a virus-infected strain of L. guyanensis. After 6 weeks of drug selection (hygromycin B), transfected parasites lacked detectable quantities of viral genomic double-stranded RNA, viral capsid protein, and RNA-dependent RNA polymerase (RDRP) activity. Evidence of viral infection was not recovered upon removal of the drug. While viral RNA transcripts were produced from electroporated expression vectors, as determined by reverse transcription-PCR, viral antigens were not detected, suggesting that the antiviral effects of hygromycin B are mediated through translation inhibition. A short-term selection study suggests that the LRV1-4 elimination may not only be a function of hygromycin B as a protein synthesis inhibitor but also possibly related to the mechanism of hygromycin B resistance in Leishmania strains.
Propagation of prion strains through specific conformers of the prion protein
Authors: Scott M R , Groth D , Tatzelt J , Torchia M , Tremblay P , DeArmond S J , Prusiner S B
Published: January 2020

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP.
The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton
Authors: Zafrullah M , Ozdener M H , Panda S K , Jameel S
Published: January 2020

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle.
A novel viral RNA species in Sindbis virus-infected cells
Authors: Wielgosz M M , Huang H V
Published: January 2020

Sindbis virus (SIN), the type alphavirus, has been studied extensively to identify the viral cis-acting sequences and proteins involved in RNA transcription and replication. However, very little is known about how these processes are coordinated. For example, synthesis of the genomic RNA and the subgenomic mRNA depends on the minus strand. Do these activities occur independently on different templates, or can replication and transcription take place simultaneously on the same template? We describe the appearance of a SIN-specific, plus-sense RNA that is intermediate in size between the genomic and subgenomic RNA species. This RNA, designated RNA II, is observed in a number of different cell lines, both early and late in infection. The number of RNA II species, their sizes, and their abundances are influenced by the subgenomic promoter. We have mapped the 3' end of RNA II to a site within the subgenomic promoter, four nucleotides before the initiation site of the subgenomic mRNA. Our results indicate that the appearance of RNA II is correlated with subgenomic mRNA transcription, such that strong or active promoters tend to increase the abundance of RNA II, relative to weak or less active promoters. RNA II is most abundantly detected with the full promoter and is at much lower abundance with the minimal promoter. The possible origins of RNA II are discussed.
The pseudorabies virus UL28 protein enters the nucleus after coexpression with the herpes simplex virus UL15 protein
Authors: Koslowski K M , Shaver P R , Wang X Y , Tenney D J , Pederson N E
Published: January 2020

Herpesvirus DNA is packaged into capsids in the nuclei of infected cells in a process requiring at least six viral proteins. Of the proteins required for encapsidation of viral DNA, UL15 and UL28 are the most conserved among herpes simplex virus type 1 (HSV), varicella-zoster virus, and equine herpesvirus 1. The subcellular distribution of the pseudorabies virus (PRV) UL28 protein was examined by in situ immunofluorescence. UL28 was present in the nuclei of infected cells; however, UL28 was limited to the cytoplasm in the absence of other viral proteins. When cells expressing variant forms of UL28 were infected with a PRV UL28-null mutant, UL28 entered the nucleus, provided the carboxyl-terminal 155 amino acids were present. Additionally, PRV UL28 entered the nucleus in cells infected with HSV. Two HSV packaging proteins were tested for the ability to affect the subcellular distribution of UL28. Coexpression of HSV UL15 enabled PRV UL28 to enter the nucleus in a manner that required the carboxyl-terminal 155 amino acids of UL28. Coexpression of HSV UL25 did not affect the distribution of UL28. We propose that an interaction between UL15 and UL28 facilitates the transport of a UL15-UL28 complex to the infected-cell nucleus.
Identification of the V1 region as a linear neutralizing epitope of the simian immunodeficiency virus SIVmac envelope glycoprotein
Authors: Jurkiewicz E , Hunsmann G , Schäffner J , Nisslein T , Lüke W , Petry H
Published: January 2020

The sequence variability of viral structure polypeptides has been associated with immune escape mechanisms. The V1 region of simian immunodeficiency virus (SIV) is a highly variable region of the SIVmac env gene. Here, we describe the V1 region as a linear neutralizing epitope. V1 region-specific neutralizing antibodies (NAb) were first demonstrated in a rabbit infected with a recombinant vaccinia virus carrying the env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben). Since we detected in this animal V1 region-specific NAb that were able to neutralize not only human immunodeficiency virus type 2 but also SIVmac32H, we investigated whether a similar immune response is evoked in macaques (Macaca mulatta) either infected with SIVmac or immunized with the external glycoprotein (gp130) of the same virus. Distinctly lower NAb titers were found in the SIVmac-infected animals than in the gp130-immunized macaques. Since the NAb titers in both groups were high enough for competition experiments, we used five overlapping peptides encompassing the whole V1 region for a detailed identification of the epitope. In each of the 12 macaques investigated, we detected a high level of NAb reacting with at least one peptide located in the central part of the V1 region. The relatively high degree of divergence, especially within the central part of the V1 region, which characterized the evolution of the retroviral sequences from the original inoculum in the infected macaques suggests the development of escape mutants. Furthermore, 3 of 12 animals developed NAb directed against the amino-terminal end of the V1 region epitope. Sequence analysis, however, revealed relatively low levels of genetic drift and genetic variability within this part of the V1 region. The induction of V1 env-specific NAb not only in gp130-immunized macaques but also in SIVmac-infected animals in combination with the increased genetic variability of this region in vivo indicates a marked biological significance of this epitope for the virus.
E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter
Authors: Pützer B M , Rumpf H , Rega S , Brockmann D , Esche H
Published: January 2020

The transformation-defective Vero cell host range mutant CS-1 of the highly oncogenic adenovirus type 12 (Ad12) (Ad12-CS-1) has a 69-bp deletion in the early region 1A (E1A) gene that removes the carboxy-terminal half of conserved region 2 and the amino-terminal half of the Ad12-specific so-called spacer that seems to play a pivotal role in the oncogenicity of the virus. Despite its deficiency in immortalizing and transforming primary rodent cells, we found that the E1A 13S protein of Ad12-CS-1 retains the ability to bind p105-RB, p107, and p130 in nuclear extract binding assays with glutathione S-transferase-E1A fusion proteins and Western blot analysis. Like wild-type E1A, the mutant protein was able to dissociate E2F from retinoblastoma-related protein-containing complexes, as judged from gel shift experiments with purified 12S and 13S proteins from transfection experiments with an E1A expression vector or from infection with the respective virus. Moreover, in transient expression assays, the 12S and 13S products of wild-type Ad12 and Ad12-CS-1 were shown to transactivate the Ad12 E1A promoter containing E2F-1 and E2F-5-motifs, respectively, in a comparable manner. The same results were obtained from transfection assays with the E2F motif-dependent E2 promoter of adenovirus type 5 or the human dihydrofolate reductase promoter. These data suggest that efficient infection by Ad12 and the correlated virus-induced reprogramming of the infected cells, including the induction of cell cycle-relevant mechanisms (e.g. E2F activation), can be uncoupled from the transformation properties of the virus.
Identification of two independent transcriptional activation domains in the Autographa californica multicapsid nuclear polyhedrosis virus IE1 protein
Authors: Slack J M , Blissard G W
Published: January 2020

The Autographa californica multicapsid nuclear polyhedrosis virus immediate-early protein, IE1, is a 582-amino-acid phosphoprotein that regulates the transcription of early viral genes. Deletion of N-terminal regions of IE1 in previous studies (G. R. Kovacs, J. Choi, L. A. Guarino, and M. D. Summers, J. Virol. 66:7429-7437, 1992) resulted in the loss of transcriptional activation, suggesting that this region may contain an acidic activation domain. To identify independently functional transcriptional activation domains, we developed a heterologous system in which potential regulatory domains were fused with a modified Escherichia coli Lac repressor protein that contains a nuclear localization signal (NLacR). Transcriptional activation by the resulting NLacR-IE1 chimeras was measured with a basal baculovirus early promoter containing optimized Lac repressor binding sites (lac operators). Chimeras containing IE1 peptides dramatically activated transcription of the basal promoter only when lac operator sequences were present. In addition, transcriptional activation by NLacR-IE1 chimeras was allosterically regulated by the lactose analog, isopropyl-beta-D-thiogalactopyranoside (IPTG). For a more detailed analysis of IE1 regulatory domains, the M1 to T266 N-terminal portion of IE1 was subdivided (on the basis of average amino acid charge) into five smaller regions which were fused in various combinations to NLacR. Regions M1 to N125 and A168 to G222 were identified as independent transcriptional activation domains. Some NLacR-IE1 chimeras exhibited retarded migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. As with wild-type IE1, this aberrant gel mobility was associated with phosphorylation. Mapping studies with the NLacR-IE1 chimeras indicate that the M1 to A168 region of IE1 is necessary for this phosphorylation-associated effect.
trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication
Authors: Lindenbach B D , Rice C M
Published: January 2020

Mutational analysis of the nonstructural protein 1 (NS1) of yellow fever virus (YF) has implicated it in viral RNA replication. To further explore this observation, we sought a method for uncoupling NS1 function from NS1 expression and processing as part of the large YF polyprotein. Here we describe a strategy for providing NS1 in trans, utilizing a noncytopathic Sindbis virus vector. Replication of a defective YF genome containing a large in-frame deletion of NS1 was dependent on functional expression of NS1. Recovered mutant virus was shown to contain the deletion and was neutralized by YF-specific antiserum. Complemented mutant virus increased in titer with kinetics similar to those of parental YF 17D but peaked at lower titers. trans-complementation has allowed us to derive high-titer, helper-free stocks of YF defective in NS1 with which to further characterize the role of this gene product in RNA replication. The first cycles of RNA replication were analyzed by using a sensitive strand-specific RNase protection assay. We document these events for mutant and wild-type viruses in the presence or absence of complementation. These data strongly suggest a role for NS1 prior to or at initial minus-strand synthesis.
Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme
Authors: Sekiguchi J , Shuman S
Published: January 2020

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.
Late treatment with polyene antibiotics can prolong the survival time of scrapie-infected animals
Authors: Demaimay R , Adjou K T , Beringue V , Demart S , Lasmézas C I , Deslys J P , Seman M , Dormont D
Published: January 2020

Amphotericin B (AmB) is one of the few drugs able to prolong survival times in experimental scrapie and delays the accumulation of PrPres, a specific marker of this disease in the brain in vivo. Previous reports showed that the AmB effect is observed only if the drug is administered around the time of infection. In the present study, intracerebrally infected mice were treated with AmB or one of its derivatives, MS-8209, between 80 and 140 days postinoculation. We observed an increased incubation time and a delay in PrPres accumulation and glial fibrillary acidic protein gene expression. Treatment starting at 80 days postinoculation was as efficient as long-term treatment starting the day of inoculation. Our results indicate that polyene antibiotics may interfere, throughout the course of the experimental disease, with the propagation of the scrapie agent.
Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1
Authors: Smith S M , Maldarelli F , Jeang K T
Published: January 2020

Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells.
An SL3-3 murine leukemia virus enhancer variant more pathogenic than the wild type obtained by assisted molecular evolution in vivo
Authors: Ethelberg S , Sørensen A B , Schmidt J , Luz A , Pedersen F S
Published: January 2020

SL3-3 is a highly T-lymphomagenic murine retrovirus in which the transcriptional enhancer is a major oncogenic determinant. Here, we describe an SL3-3 enhancer variant that induced T-cell lymphomas in all inoculated mice with a shorter latency period than wild-type SL3-3. The enhancer repeat region of this variant contains two deletions encompassing the nuclear factor 1 binding sites in addition to an additional intact enhancer repeat element. Tumors induced by this variant were T-cell lymphomas, as indicated by T-cell receptor rearrangements, and contained the input provirus enhancer regions. The variant was the result of mutation of specific transcription factor binding sites in the viral enhancer, isolation of rare second-site enhancer variants from the resulting induced tumors, and subsequent restoration of the original first-site mutations of one such variant. We have termed this process assisted molecular evolution.
Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding
Authors: Verdaguer N , Fita I , Domingo E , Mateu M G
Published: January 2020

Neutralization of an aphthovirus by monovalent binding of an antibody is reported. Foot-and-mouth disease virus (FMDV) clone C-S8c1 was neutralized by monoclonal antibody (MAb) SD6, which was directed to a continuous epitope within a major antigenic site of the G-H loop of capsid protein VP1. On a molar basis, the Fab fragment was at most fivefold less active in neutralization than the intact antibody, and both blocked virus attachment to cells. Neither the antibody nor the Fab fragment caused aggregation of virions, as evidenced by sucrose gradient sedimentation studies of the antibody-virus complex formed at antibody to virion ratios of 1:50 to 1:10,000. The results of neutralization of infectivity and of ultracentrifugation are fully consistent with structural data based on X-ray crystallographic and cryoelectron microscopy studies, which showed monovalent interaction of the antibody with a critical receptor binding motif Arg-Gly-Asp. The conclusions of these neutralization studies are that (i) bivalent binding of antibody is not a requisite for strong neutralization of aphthoviruses and (ii) aggregation of viral particles, which has been proposed to be the dominant neutralization mechanism of antibodies that bind monovalently to virions, is not necessary for the neutralization of FMDV C-S8c1 by MAb SD6.
Reduced viral load and lack of CD4 depletion in SCID-hu mice infected with Rev-independent clones of human immunodeficiency virus type 1
Authors: Valentin A , Aldrovandi G , Zolotukhin A S , Cole S W , Zack J A , Pavlakis G N , Felber B K
Published: January 2020

The posttranscriptional control element CTE of the simian type D retrovirus has been shown to support replication of Rev-Rev-responsive-element (RRE)-deficient molecular clones of human immunodeficiency virus type 1 (HIV-1). Upon infection of peripheral blood mononuclear cells in vitro, these CTE-containing Rev-independent viruses that are nef+ or nef-minus showed lower replicative capacity and infectivity than the wild-type HIV-1. We studied the effects of Rev-RRE replacement by the CTE on HIV-1 expression with SCID-hu mice. The nef+ and nef-minus Rev-independent viruses established infection with kinetics slower than that of the nef-minus NL4-3. Most importantly, no depletion of CD4-bearing thymocytes was observed after 6 weeks for mice infected with these Rev-independent viruses. This is in contrast to the infection with both wild-type and nef-minus viruses, which led to varying depletion of thymocytes. These data suggest an attenuated phenotype for growth and cytotoxicity of the Rev-independent HIV-1 clones in SCID-hu mice, independent of the presence of Nef. The mutant viruses, which have the essential Rev-RRE regulatory system eliminated, display a distinct phenotype not previously observed with HIV mutant viruses having deletions of accessory genes. Therefore, replacement of the Rev-RRE regulatory axis may generate viruses with altered biological properties in vivo.
Functional identification of nucleotides conferring substrate specificity to retroviral integrase reactions
Authors: Balakrishnan M , Jonsson C B
Published: January 2020

The long terminal repeats (LTRs) that flank the retroviral DNA genome play a distinct role in the integration process by acting as specific substrates for the integrase (IN). The role of LTR sequences in providing substrate recognition and specificity to integration reactions was investigated for INs from human immunodeficiency virus type 1 (HIV-1), Moloney murine leukemia virus (M-MuLV), human T-cell leukemia virus type 1 (HTLV-1), and human T-cell leukemia virus type 2 (HTLV-2). Overall, these INs required specific LTR sequences for optimal catalysis of 3'-processing reactions, as opposed to strand transfer and disintegration reactions. It is of particular note that in strand transfer reactions the sites of integration were similar among the four INs. In the 3'-processing reaction, sequence specificity for each IN was traced to the three nucleotides proximal to the conserved CA. Reactions catalyzed by M-MuLV IN were additionally influenced by upstream regions. The nucleotide requirements for optimal catalysis differed for each IN. HIV-1 IN showed a broad range of substrate specificities, while HTLV-1 IN and HTLV-2 IN had more defined sequence requirements. M-MuLV IN exhibited greater activity with the heterologous LTR substrates than with its own wild-type substrate. This finding was further substantiated by the high levels of activity catalyzed by the IN on modified M-MuLV LTRs. This work suggests that unlike the other INs examined, M-MuLV IN has evolved with an IN-LTR interaction that is suboptimal.
Direct ex vivo simian immunodeficiency virus (SIV)-specific cytotoxic activity detected from small intestine intraepithelial lymphocytes of SIV-infected macaques at an advanced stage of infection
Authors: Couëdel-Courteille A , Le Grand R , Tulliez M , Guillet J G , Venet A
Published: January 2020

Human immunodeficiency virus (HIV) induces a profound disorganization of the lymphoid tissues with marked abnormalities of the immune system at the terminal stage of infection. Since the digestive mucosal immune system is by far the largest lymphoid organ of the body, we attempted to evaluate its functional activity in advanced stages of simian immunodeficiency virus (SIV) infection in the SIV-macaque model of HIV infection. Two chronically intravenously SIV-infected macaques, including one at the AIDS stage, were studied. Intestinal intraepithelial lymphocytes (IEL) were isolated, analyzed, and compared to lymphocytes obtained from blood, spleen, and different lymph nodes: IEL were predominantly CD8+ T lymphocytes expressing the alphaE beta7 integrin and lacking the CD28 coactivatory molecule. A direct ex vivo SIV-specific cytotoxic activity was prominently found in the IEL of both macaques and was weaker or absent in the other sites. To our knowledge, this is the first report of SIV-specific cytotoxic activity from small intestine IEL in SIV-infected macaques. Considering the high similitude of the SIV-macaque model with the HIV infection in humans, these results may be highly important for the pathogenesis of HIV infection and more generally important for the characterization and function of digestive CD8+ IEL population.
Phenotypic and genetic analysis of Lymantria dispar nucleopolyhedrovirus few polyhedra mutants: mutations in the 25K FP gene may be caused by DNA replication errors
Authors: Bischoff D S , Slavicek J M
Published: January 2020

We previously demonstrated that polyhedron formation (PF) mutants arise at a high frequency during serial passage of the Lymantria dispar nucleopolyhedrovirus (LdMNPV) in the L. dispar 652Y cell line (J. M. Slavicek, N. Hayes-Plazolles, and M. E. Kelly, Biol. Control 5:251-261, 1995). Most of these PF mutants exhibited the traits of few polyhedra (FP) mutants; however, no large DNA insertions or deletions that correlated with the appearance of the FP phenotype were found. In this study, we have characterized several of the PF mutants at the phenotypic and genetic levels. Genetic techniques were used to group the mutations in the LdMNPV PF mutants to the same or closely linked genes. Wild-type viruses were recovered after coinfection of L. dispar 652Y cells with certain combinations of PF mutants. These viruses were analyzed by restriction endonuclease analysis and found to be chimeras of the original PF mutants used in the coinfections. Marker rescue experiments localized the mutations in one group of PF isolates to the region containing the LdMNPV 25K FP gene. The mutations in these PF mutants were identified. Four of five of the LdMNPV FP mutants contain small insertions or deletions within the 25K FP gene. The fifth LdMNPV FP mutant analyzed contained a large deletion that truncated the C terminus of the 25K FP gene product. All of the deletions occurred within the same potential hairpin loop structure, which had the lowest free energy value (most stable hairpin) of the five potential hairpin loop structures present in the 25K FP gene. One of the insertion mutants contained an extra base within a repetitive sequence. These types of mutations are likely caused by errors that occur during DNA replication. The relationship between the types of mutations found within the LdMNPV 25K FP gene and DNA replication-based mutagenesis is discussed.
A novel terminal resolution-like site in the adeno-associated virus type 2 genome
Authors: Wang X S , Srivastava A
Published: January 2020

The adeno-associated virus 2 (AAV) contains a single-stranded DNA genome of which the terminal 145 nucleotides are palindromic and form T-shaped hairpin structures. These inverted terminal repeats (ITRs) play an important role in AAV DNA replication and resolution, since each of the ITRs contains a terminal resolution site (trs) that is the target site for the AAV rep gene products (Rep). However, the Rep proteins also interact with the AAV DNA sequences that lie outside the ITRs, and the ITRs also play a crucial role in excision of the proviral genome from latently infected cells or from recombinant AAV plasmids. To distinguish between Rep-mediated excision of the viral genome during rescue from recombinant AAV plasmids and the Rep-mediated resolution of the ITRs during AAV DNA replication, we constructed recombinant AAV genomes that lacked either the left or the right ITR sequence and one of the Rep-binding sites (RBSs). No rescue and replication of the AAV genome occurred from these plasmids following transfection into adenovirus type 2-infected human KB cells, as expected. However, excision and abundant replication of the vector sequences was clearly detected from the plasmid that lacked the AAV left ITR, suggesting the existence of an additional putative excision site in the left end of the AAV genome. This site was precisely mapped to one of the AAV promoters at map unit 5 (AAV p5) that also contains an RBS. Furthermore, deletion of this RBS abolished the rescue and replication of the vector sequences. These studies suggest that the Rep-mediated cleavage at the RBS during viral DNA replication may, in part, account for the generation of the AAV defective interfering particles.
Subcellular compartmentalization of adeno-associated virus type 2 assembly
Authors: Wistuba A , Kern A , Weger S , Grimm D , Kleinschmidt J A
Published: January 2020

Using immunofluorescence and in situ hybridization techniques, we studied the intracellular localization of adeno-associated virus type 2 (AAV-2) Rep proteins, VP proteins, and DNA during the course of an AAV-2/adenovirus type 2 coinfection. In an early stage, the Rep proteins showed a punctate distribution pattern over the nuclei of infected cells, reminiscent of replication foci. At this stage, no capsid proteins were detectable. At later stages, the Rep proteins were distributed more homogeneously over the nuclear interior and finally became redistributed into clusters slightly enriched at the nuclear periphery. During an intermediate stage, they also appeared at an interior part of the nucleolus for a short period, whereas most of the time the nucleoli were Rep negative. AAV-2 DNA colocalized with the Rep proteins. All three capsid proteins were strongly enriched in the nucleolus in a transient stage of infection, when the Rep proteins homogeneously filled the nucleoplasm. Thereafter, they became distributed over the whole nucleus and colocalized in nucleoplasmic clusters with the Rep proteins and AAV-2 DNA. While VP1 and VP2 strongly accumulated in the nucleus, VP3 was almost equally distributed between the nucleus and cytoplasm. Capsids, visualized by a conformation-specific antibody, were first detectable in the nucleoli and then spread over the whole nucleoplasm. This suggests that nucleolar components are involved in initiation of capsid assembly whereas DNA packaging occurs in the nucleoplasm. Expression of a transfected full-length AAV-2 genome followed by adenovirus infection showed all stages of an AAV-2/adenovirus coinfection, whereas after expression of the cap gene alone, capsids were restricted to the nucleoli and did not follow the nuclear redistribution observed in the presence of the whole AAV-2 genome. Coexpression of Rep proteins released the restriction of capsids to the nucleolus, suggesting that the Rep proteins are involved in nuclear redistribution of AAV capsids during viral infection. Capsid formation was dependent on the concentration of expressed capsid protein.
A novel cellular site-specific DNA-binding protein cooperates with the viral NS1 polypeptide to initiate parvovirus DNA replication
Authors: Christensen J , Cotmore S F , Tattersall P
Published: January 2020

Replication of linear single-stranded parvovirus DNA proceeds by a rolling-hairpin mechanism which generates long, palindromic, duplex concatamers. Processing to monomer length requires initiation from origins of DNA replication located at the 3' and 5' ends of each embedded monomer, reactions which can be recapitulated in vitro for minute virus of mice (MVM). To determine which cellular proteins were essential for replication from these origins, S100 extracts from 293S cells were fractionated on phosphocellulose. When recombined, these fractions were able to support replication in vitro, dependent on the viral initiator protein NS1, using plasmid forms of the 5' origin or the minimal 3' origin as templates. Fraction P-cell 1 contains two factors, replication protein A (RPA) and proliferating-cell nuclear antigen (PCNA), known to be essential for simian virus 40 replication in vitro. When P-cell 1 was replaced with purified recombinant RPA and PCNA, NS1-mediated MVM replication initiated from the 5' origin but not from the 3' origin. The 3' origin is a 50-bp sequence containing three distinct recognition elements, an NS1 binding site, a site at which NS1 nicks the DNA to generate the priming 3' OH, and a region containing a consensus activated transcription factor (ATF) binding site. To identify the missing factor(s) for 3' origin replication, P-cell 1 was fractionated by further chromatography and active fractions were identified by their ability to complement RPA, PCNA, and P-cell 2 for NS1-mediated, origin-specific replication. Gel shift and UV cross-linking analysis of the replication-competent fractions revealed a novel 110-kDa sequence-specific DNA binding protein which recognized the consensus ATF binding site region of the origin and which we have termed parvovirus initiation factor, or PIF. Binding of PIF appears to activate the endonuclease function of NS1, allowing efficient and specific nicking of the 3' minimal origin under stringent conditions in vitro.
Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection
Authors: Stephensen C B , Welter J , Thaker S R , Taylor J , Tartaglia J , Paoletti E
Published: January 2020

Canine distemper virus (CDV) infection of ferrets causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system (CNS). We have tested candidate CDV vaccines incorporating the fusion (F) and hemagglutinin (HA) proteins in the highly attenuated NYVAC strain of vaccinia virus and in the ALVAC strain of canarypox virus, which does not productively replicate in mammalian hosts. Juvenile ferrets were vaccinated twice with these constructs, or with an attenuated live-virus vaccine, while controls received saline or the NYVAC and ALVAC vectors expressing rabies virus glycoprotein. Control animals did not develop neutralizing antibody and succumbed to distemper after developing fever, weight loss, leukocytopenia, decreased activity, conjunctivitis, an erythematous rash typical of distemper, CNS signs, and viremia in peripheral blood mononuclear cells (as measured by reverse transcription-PCR). All three CDV vaccines elicited neutralizing titers of at least 1:96. All vaccinated ferrets survived, and none developed viremia. Both recombinant vaccines also protected against the development of symptomatic distemper. However, ferrets receiving the live-virus vaccine lost weight, became lymphocytopenic, and developed the erythematous rash typical of CDV. These data show that ferrets are an excellent model for evaluating the ability of CDV vaccines to protect against symptomatic infection. Because the pathogenesis and clinical course of CDV infection of ferrets is quite similar to that of other Morbillivirus infections, including measles, this model will be useful in testing new candidate Morbillivirus vaccines.
Role of early and late replication events in induction of apoptosis by baculoviruses
Authors: LaCount D J , Friesen P D
Published: January 2020

Autographa californica nuclear polyhedrosis virus (AcMNPV) mutants that lack the apoptotic suppressor gene p35 cause apoptosis in Spodoptera frugiperda SF21 cells. To identify a viral signal(s) that induces programmed cell death, we first defined the timing of apoptotic events during infection. Activation of a P35-inhibitable caspase, intracellular fragmentation of host and AcMNPV DNA, and cell membrane blebbing coincided with the initiation of viral DNA synthesis between 9 and 12 h after infection and thus suggested that apoptotic signaling begins at or before this time. Virus entry was required since binding of budded virus to host cell receptors alone was insufficient to induce apoptosis. To therefore determine the contribution of early and late replication events to apoptotic signaling, we used the AcMNPV mutant ts8 with a temperature-sensitive lesion in the putative helicase gene p143. At the nonpermissive temperature at which viral DNA synthesis was conditionally blocked, ts8 caused extensive apoptosis of the SF21 cell line p3576D, which dominantly interferes with anti-apoptotic function of viral P35. Confirming that apoptosis can be induced in the absence of normal viral DNA synthesis, parental SF21 cells also underwent apoptosis when infected with a ts8 p35 deletion mutant at the nonpermissive temperature. However, maximum levels of ts8 p35 deletion mutant-induced apoptosis required a temperature-sensitive event(s) that included the initiation of viral DNA synthesis. Collectively, these data suggested that baculovirus-induced apoptosis can be triggered by distinct early (pre-DNA synthesis) and late replicative events, including viral DNA synthesis or late gene expression.
bcl-2 acts early to restrict Semliki Forest virus replication and delays virus-induced programmed cell death
Authors: Scallan M F , Allsopp T E , Fazakerley J K
Published: January 2020

As characterized by morphological assessment and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, Semliki Forest virus (SFV) infection of rat prostatic adenocarcinoma cells triggers an apoptotic cell response. Cell death proceeded more rapidly following infection with the neurovirulent L10 strain of SFV than with the avirulent A7 strain. Overexpression of the antiapoptotic proto-oncogene bcl-2 allowed survival of cultures infected with either strain of virus. bcl-2 overexpression drastically reduced the numbers of productively infected cells within the cultures. In situ hybridization for viral message-sense RNA coupled with immunostaining for viral protein indicated that bcl-2 functions at an early stage of the virus life cycle, at entry, pretranscriptional events or at transcription, to inhibit virus replication. Double-immunofluorescent labeling for bcl-2 and viral glycoproteins revealed double-positive cells, demonstrating that with time, this early block in replication can be overcome. These productively infected bcl-2-expressing cells do, with time, undergo apoptosis. As a result of changing the balance between cell death and cell division by restricting productive virus replication and delaying virus-induced cell death, bcl-2 expression led to the establishment of chronically infected cell lines which could be passaged.
A molecularly cloned, pathogenic, neutralization-resistant simian immunodeficiency virus, SIVsmE543-3
Authors: Hirsch V , Adger-Johnson D , Campbell B , Goldstein S , Brown C , Elkins W R , Montefiori D C
Published: January 2020

An infectious molecular clone of simian immunodeficiency virus SIVsm was derived from a biological isolate obtained late in disease from an immunodeficient rhesus macaque (E543) with SIV-induced encephalitis. The molecularly cloned virus, SIVsmE543-3, replicated well in macaque peripheral blood mononuclear cells and monocyte-derived macrophages and resisted neutralization by heterologous sera which broadly neutralized genetically diverse SIV variants in vitro. SIVsmE543-3 was infectious and induced AIDS when inoculated intravenously into pig-tailed macaques (Macaca nemestrina). Two of four infected macaques developed no measurable SIV-specific antibody and succumbed to a wasting syndrome and SIV-induced meningoencephalitis by 14 and 33 weeks postinfection. The other two macaques developed antibodies reactive in Western blot and virus neutralization assays. One macaque was sacrificed at 1 year postinoculation, and the survivor has evidence of immunodeficiency, characterized by persistently low CD4 lymphocyte subsets in the peripheral blood. Plasma samples from these latter animals neutralized SIVsmE543-3 but with much lower efficiency than neutralization of other related SIV strains, confirming the difficulty by which this molecularly cloned virus is neutralized in vitro. SIVsmE543-3 will provide a valuable reagent for studying SIV-induced encephalitis, mapping determinants of neutralization, and determining the in vivo significance of resistance to neutralization in vitro.
Macrophage tropism of human immunodeficiency virus type 1 and utilization of the CC-CKR5 coreceptor
Authors: Cheng-Mayer C , Liu R , Landau N R , Stamatatos L
Published: January 2020

The recent identification of the CC-CKR5 beta chemokine receptor as a major cofactor for entry of macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1) raises the question of whether macrophage tropism is determined by utilization of this chemokine receptor. We observe that in addition to macrophage-tropic isolates of clades A, B, and E, macrophage-tropic isolates of clade F also utilize the CC-CKR5 molecule for entry. However, using single-round replication-competent reporter viruses carrying the envelope genes of T-cell line-tropic or macrophage-tropic phenotypic recombinant and mutant HIV-1 strains in infection of stable cell lines that coexpress the CD4 and chemokine receptors, we were unable to establish a strict correlation between macrophage tropism and utilization of the CC-CKR5 chemokine receptor. This latter finding suggests that a cofactor other than CC-CKR5 serves to determine entry into primary macrophages.
Structure of replicating hepatitis C virus (HCV) quasispecies in the liver may not be reflected by analysis of circulating HCV virions
Authors: Cabot B , Esteban J I , Martell M , Genescà J , Vargas V , Esteban R , Guardia J , Gómez J
Published: January 2020

We have analyzed the population of hepatitis C virus (HCV) sequences in paired liver and serum samples from four patients with chronic hepatitis C. Sequences from three different biopsy specimens from a liver explant from one patient were compared with each other and with the circulating sequences. Our results demonstrate that the circulating quasispecies does not necessarily reflect the viral population replicating in the liver and that this is not due to a macroscopic anatomic compartmentalization of HCV replication. This finding has important implications for the pathogenesis and natural history of chronic HCV infection.
An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis
Authors: Myers T M , Moyer S A
Published: January 2020

The nucleocapsid protein (NP) of Sendai virus encapsidates the genome RNA, forming a helical nucleocapsid which is the template for RNA synthesis by the viral RNA polymerase. The NP protein is thought to have both structural and functional roles, since it is an essential component of the NP0-P (P, phosphoprotein), NP-NP, nucleocapsid-polymerase, and RNA-NP complexes required during viral RNA replication. To identify domains in the NP protein, mutants were constructed by using clustered charge-to-alanine mutagenesis in a highly charged region from amino acids 107 to 129. Each of the mutants supported RNA encapsidation in vitro. The product nucleocapsids formed with three mutants, NP114, NP121, and NP126, however, did not serve as templates for further amplification in vivo, while NP107, NP108, and NP111 were nearly like wild-type NP in vivo. This template defect in the NP mutants from amino acids 114 to 129 was not due to a lack of NP0-P, NP-NP, or nucleocapsid-polymerase complex formation, since these interactions were normal in these mutants. We propose that amino acids 114 to 129 of the NP protein are required for the nucleocapsid to function as a template in viral genome replication.
Herpes simplex virus immediate-early proteins ICP0 and ICP4 activate the endogenous human alpha-globin gene in nonerythroid cells
Authors: Cheung P , Panning B , Smiley J R
Published: January 2020

Globin genes are normally expressed only in erythroid cell lineages. However, we found that the endogenous alpha-globin gene is activated following infection of human fibroblasts and HeLa cells with herpes simplex virus (HSV), leading to accumulation of correctly initiated transcripts driven by the alpha-globin promoter. The alpha1- and alpha2-globin genes were both induced, but expression of beta- or zeta-globin genes could not be detected. Experiments using HSV mutants showed that null mutations in the genes encoding the viral immediate-early proteins ICP4 and ICP22 reduced induction approximately 10-fold, while loss of ICP0 function had a smaller inhibitory effect. Transient transfection experiments showed that ICP0 and ICP4 are each sufficient to trigger detectable expression of the endogenous gene, while ICP22 had no detectable effect in this assay. ICP4 also strongly enhanced expression of transfected copies of the alpha2-globin gene. In contrast, the adenovirus E1a protein did not activate the endogenous gene and inhibited expression of the plasmid-borne alpha2-globin gene. Previous studies have led to the hypothesis that chromosomal alpha-globin genes are subject to chromatin-dependent repression mechanism that prevents expression in nonerythroid cells. Our data suggest that HSV ICP0 and ICP4 either break or bypass this cellular gene silencing mechanism.