bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins
Authors: Yang Q , Chen F , Trempe J P
Published: January 2020

The replication (rep) gene of adeno-associated virus (AAV) is involved in AAV DNA replication, gene regulation, and inhibition of cellular transformation induced by various oncogenes. To study the rep gene's antiproliferative effects, we have developed cell lines which express the replication proteins under the control of an inducible mouse metallothionein transcription promoter. The Rep78 protein produced in these cell lines binds to the AAV terminal repeat sequences in vitro and supports AAV DNA replication and trans activation of the AAV p40 transcription promoter in vivo. These cell lines are capable of assembling infectious viruses containing a mutant rep gene or a vector bearing a heterologous gene. Growth rate and colony formation efficiency assays indicated that rep gene expression substantially altered cellular proliferation. Long-term induction of the cell lines followed by removal of the inducing agent suggested that constitutive expression of the Rep proteins does not necessarily result in cell death and that the cells can recover from the cytostatic effects. Flow cytometry analysis indicated that the presence of the Rep proteins increased the population of cells in the S phase of the cell cycle. Thus the rep gene's antiproliferative effects may be realized by interference with cellular DNA replication.
Incorporation of homologous and heterologous proteins into the envelope of Moloney murine leukemia virus
Authors: Suomalainen M , Garoff H
Published: January 2020

The efficiencies with which homologous and heterologous proteins are incorporated into the envelope of Moloney murine leukemia virus (M-MuLV) have been analyzed by utilizing a heterologous, Semliki Forest virus-driven M-MuLV assembly system and quantitative pulse-chase assays. Homologous M-MuLV spike protein was found to be efficiently incorporated into extracellular virus particles when expressed at a relatively low density at the plasma membrane. In contrast, efficient incorporation of heterologous proteins (the spike complex of Semliki Forest virus and a cytoplasmically truncated mutant of the human transferrin receptor) was observed only when these proteins were expressed at high densities at the cell surface. These results imply that homologous and heterologous proteins are incorporated into the M-MuLV envelope via two distinct pathways.
Interaction of herpes simplex virus type 1 DNA polymerase and the UL42 accessory protein with a model primer template
Authors: Gottlieb J , Challberg M D
Published: January 2020

Genetic and biochemical studies have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication. A number of studies have previously suggested that these two proteins specifically interact, and more recent studies have confirmed that the viral DNA polymerase from HSV-1-infected cells consists of a heterodimer of the UL30 (Pol; the catalytic subunit) and UL42 polypeptides. A comparison of the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells indicated that the Pol-UL42 complex is more highly processive than Pol alone on singly primed M13 single-stranded substrates. The results of these studies are consistent with the idea that the UL42 polypeptide is an accessory subunit of the HSV-1 DNA polymerase that acts to increase the processivity of polymerization. Preliminary experiments suggested that the increase in processivity was accompanied by an increase in the affinity of the polymerase for the ends of linear duplex DNA. We have further characterized the effect of the UL42 polypeptide on a defined hairpin primer template substrate. Gel shift and filter binding studies show that the affinity of the Pol catalytic subunit for the 3' terminus of the primer template increases 10-fold in the presence of UL42. DNase I footprinting experiments indicate that the Pol catalytic subunit binds to the primer template at a position that protects 14 bp of the 3' duplex region and an adjacent 18 bases of the single-stranded template. The presence of the UL42 polypeptide results in the additional protection of a contiguous 5 to 14 bp in the duplex region but does not affect the 5' position of the Pol subunit. Free UL42 protects the entire duplex region of the substrate but does not bind to the single-stranded region. Taken together, these results suggest that the increase in processivity in the presence of UL42 is related to the double-stranded DNA-binding activity of free UL42 and that the role of UL42 in the DNA polymerase complex is to act as a clamp, decreasing the probability that the polymerase will dissociate from the template after each cycle of catalysis.
Three-dimensional structure of baculovirus-expressed Norwalk virus capsids
Authors: Prasad B V , Rothnagel R , Jiang X , Estes M K
Published: January 2020

The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty capsid, 38.0 nm in diameter, exhibits T = 3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the capsid protein and large hollows at the icosahedral 5- and 3-fold axes. Despite its distinctive architecture, the Norwalk virus capsid has several similarities with the structures of T = 3 single-stranded RNA (ssRNA) viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (P1), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 mm. It is suspected that the hinge peptide in the adjoining region between the central domain and the shell domain may facilitate the subunits adapting to various quasi-equivalent environments. Architectural similarities between the Norwalk virus capsid and the other ssRNA viruses have suggested a possible domain organization along the primary sequence of the Norwalk virus capsid protein. It is suggested that the N-terminal 250 residues constitute the lower shell domain (S) with an eight-strand beta-barrel structure and that the C-terminal residues beyond 250 constitute the protruding (P1+P2) domains. A lack of an N-terminal basic region and the ability of the Norwalk virus capsid protein to form empty T = 3 shells suggest that the assembly pathway and the RNA packing mechanisms may be different from those proposed for tomato bushy stunt virus and southern bean mosaic virus but similar to that in tymoviruses and comoviruses.
Development of the anti-gp120 antibody response during seroconversion to human immunodeficiency virus type 1
Authors: Moore J P , Cao Y , Ho D D , Koup R A
Published: January 2020

We have studied the development of the antibody response to the surface glycoprotein gp120 of human immunodeficiency virus type 1 in three individuals who presented with primary human immunodeficiency virus type 1 infection syndrome. Serum anti-gp120 antibodies were first detected 4 to 23 days after presentation, after p24 antigen and infectious-virus titers in the peripheral blood had declined manyfold from their highest values. Whether anti-gp120 antibodies present at undetectable levels are involved in clearance of viremia remains unresolved. Among the earliest detectable anti-gp120 antibodies were those to conformationally sensitive epitopes; these antibodies were able to block the binding of gp120 monomers to soluble CD4 or to a human monoclonal antibody to a discontinuous epitope overlapping the CD4-binding site. Some of these antibodies were type specific to a degree, in that they were more effective at blocking ligand binding to autologous gp120 than to heterologous gp120. However, the appearance of these antibodies did not correlate with that of antibodies able to neutralize the autologous virus in vitro by a peripheral blood mononuclear cell-based assay. Antibodies to the V3 loop were detected at about the same time as, or slightly later than, those to the CD4-binding site. There was a weak correlation between the presence of antibodies to the V3 loop and autologous virus-neutralizing activity in two of three individuals studied. However, serum from the third individual contained V3 antibodies but lacked the ability to neutralize the autologous virus in vitro, even immediately after seroconversion. Thus, no simple, universal correlate of autologous virus-neutralizing activity in a peripheral blood mononuclear cell-based assay is apparent from in vitro assays that rely on detecting antibody interactions with monomeric gp120 or fragments thereof.
Transcriptional activation by the human cytomegalovirus immediate-early proteins: requirements for simple promoter structures and interactions with multiple components of the transcription complex
Authors: Lukac D M , Manuppello J R , Alwine J C
Published: January 2020

We have utilized a number of well-defined, simple, synthetic promoters (upstream factor binding sites and TATA elements) to analyze the activation mechanisms of the human cytomegalovirus immediate-early (IE) proteins. We found that the 86-kDa IE protein (known as IEP86, IE2(559aa), or ppUL122a) can recognize and activate a variety of simple promoters, in agreement with the observation that it is a promiscuous activator. However, in the comparison of otherwise identical promoters IEP86 does have preferences for specific TATA elements (hsp70 > adenovirus E2 > simian virus 40 early) and specific upstream transcription factor binding sites (CAAT > SP1 approximately Tef-1 > ATF; no activation with AP1 or OCT). In contrast, the 72-kDa IE protein (known as IEP72, IE1(491aa), or ppUL123) alone did not significantly activate the simple promoters under our experimental conditions. However, each promoter activated by IEP86 was synergistically affected by the addition of IEP72. In addition, the 55-kDa IE protein (IEP55, a splice variant form of IE2, IE2(425aa), or ppUL122b) repeatedly had a negative effect, downregulating the activation of promoters caused by IEP86 and the synergy of IEP86 and IEP72. We show that the ability of IEP86 to activate many simple promoters correlates not only with its previously described ability to interact with the TATA-binding protein (TBP) (B. A. Furnari, E. Poma, T. F. Kowalik, S.-M. Huong, and E.-S. Huang, J. Virol. 67:4981-4991, 1993; C. Hagemeier, S. Walker, R. Caswell, T. Kouzarides, and J. Sinclair, J. Virol. 66:4452-4456, 1992; R. Jupp, S. Hoffman, R. M. Stenberg, J. A. Nelson, and P. Ghazal, J. Virol. 67:7539-7546, 1993) but also with its ability to interact with the transcription factors which bind to the upstream element of promoters it activated (e.g., SP1 and Tef-1 but not Oct-1). This ability to have multiple interactions with the promoter complex may be crucial for transcriptional activation, since the IE proteins cannot activate promoters having only a TATA element or only an upstream transcription factor binding site. In addition, we show that proteins which bind IEP86 also bind to IEP55. Thus, the negative effect on transcription noted with IEP55 may be the result of competition with IEP86 for interaction with the promoter complex. The synergy caused by IEP72 appears to be mediated by a more indirect mechanism. This is suggested by our observation that IEP72 could not bind to any of the proteins tested (TBP, Tef-1, or Oct-1) or to IEP86.
Phenotype of the herpes simplex virus type 1 protease substrate ICP35 mutant virus
Authors: Matusick-Kumar L , Hurlburt W , Weinheimer S P , Newcomb W W , Brown J C , Gao M
Published: January 2020

The herpes simplex virus type 1 ICP35 assembly protein is involved in the formation of viral capsids. ICP35 is encoded by the UL26.5 gene and is specifically processed by the herpes simplex virus type 1 protease encoded by the UL26 gene. To better understand the functions of ICP35 in infected cells, we have isolated and characterized an ICP35 mutant virus, delta ICP35. The mutant virus was propagated in complementing 35J cells, which express wild-type ICP35. Phenotypic analysis of delta ICP35 shows that (i) mutant virus growth in Vero cells was severely restricted, although small amounts of progeny virus was produced; (ii) full-length ICP35 protein was not produced, although autoproteolysis of the protease still occurred in mutant-infected nonpermissive cells; (iii) viral DNA replication of the mutant proceeded at wild-type levels, but only a very small portion of the replicated DNA was processed to unit length and encapsidated; (iv) capsid structures were observed in delta ICP35-infected Vero cells by electron microscopy and by sucrose sedimentation analysis; (v) assembly of VP5 into hexons of the capsids was conformationally altered; and (vi) ICP35 has a novel function which is involved in the nuclear transport of VP5.
Effects of natural sequence variation on recognition by monoclonal antibodies neutralize simian immunodeficiency virus infectivity
Authors: Choi W S , Collignon C , Thiriart C , Burns D P , Stott E J , Kent K A , Desrosiers R C
Published: January 2020

The determinants of immune recognition by five monoclonal antibodies (KK5, KK9, KK17, Senv7.1, and Senv101.1) that neutralize simian immunodeficiency virus infectivity were analyzed. These five neutralizing monoclonal antibodies were generated to native SIVmac251 envelope glycoprotein expressed by a vaccinia virus recombinant vector. All five recognize conformational or discontinuous epitopes and require native antigen for optimal recognition. These monoclonal antibodies also recognize SIVmac239 gp120, but they do not recognize gp120 of two natural variants of SIVmac239, 1-12 and 8-22, which evolved during the course of persistent infection in vivo (D.P.W. Burns and R.C. Desrosiers, J. Virol. 65:1843-1854, 1991). Recombinant viruses which were constructed by exchanging variable regions between SIVmac239 and variant 1-12 were used to define domains important for recognition. Radioimmunoprecipitation analysis demonstrated that sequence changes in variable regions 4 and 5 (V4/V5) were primarily responsible for the loss of recognition of the 1-12 variant. Site-specific mutants were used to define precise changes that eliminate recognition by these neutralizing antibodies. Changing N-409 to D, deletion of KPKE, and deletion of KEQH in V4 each resulted in loss of recognition by all five monoclonal antibodies. SIVs with these natural sequence changes are still replication competent and viable. Changing A-417 to T or A/N-417/418 to TK in V4 or Q-477 to K in V5 did not alter recognition detectably. These results define specific, naturally occurring sequence changes in V4 of SIVmac that result in loss of recognition by one class of SIVmac neutralizing antibodies.
The p15gag and p12gag regions are both necessary for the pathogenicity of the murine AIDS virus
Authors: Kubo Y , Kakimi K , Higo K , Wang L , Kobayashi H , Kuribayashi K , Masuda T , Hirama T , Ishimoto A
Published: January 2020

The defective murine AIDS (MAIDS) virus has unique sequences in its p15gag and p12gag regions. To clarify whether these sequences are responsible for the development of MAIDS, we constructed recombinant viruses by replacing various regions of the gag gene of the nonpathogenic replication-competent LP-BM5 ecotropic virus with those of the MAIDS virus. Recombinants containing both unique sequences of the MAIDS virus were replication defective and induced MAIDS. However, a recombinant containing either the p15gag or p12gag region of the MAIDS virus was also replication defective but nonpathogenic in mice. A recombinant virus containing only the p30gag region of the MAIDS virus was replication competent and nonpathogenic. These results indicate that the p15gag and p12gag regions of the MAIDS virus do not function like those of replication-competent viruses and that both of the unique sequences in the p15gag and p12gag regions are required to develop MAIDS.
Myristylation of Pr60gag of the murine AIDS-defective virus is required to induce disease and notably for the expansion of its target cells
Authors: Huang M , Jolicoeur P
Published: January 2020

Murine AIDS (MAIDS) is characterized by severe lymphadenopathy and splenomegaly. The proliferation of the infected target B cells is also an important manifestation of the disease (M. Huang, C. Simard, D. G. Kay, and P. Jolicoeur, J. Virol. 65:6562-6571, 1991). The etiologic agent of MAIDS is a defective murine leukemia virus that is deleted of most of its pol and env genes and appears to encode a single protein, the Gag precursor Pr60gag protein. Pr60gag is myristylated and attached to the plasma membrane. To study the role myristylation on the function of Pr60gag, we have generated a myristylation-negative (Myr-) mutant of the MAIDS defective virus. We found that Myr- Pr60gag interacted less tightly with the plasma membrane. In addition, the Myr- MAIDS defective virus mutant was unable to induce expansion of infected cells and was nonpathogenic. These results emphasize the essential role of Pr60gag in the disease process. Our data also suggest that Pr60gag, once recruited to the cell membrane through its myristylation, interacts with other membrane-bound effectors to send signals to induce proliferation of the infected cells and to initiate immune dysfunctions.
Induction in vitro of primary cytotoxic T-lymphocyte responses with DNA encoding herpes simplex virus proteins
Authors: Rouse R J , Nair S K , Lydy S L , Bowen J C , Rouse B T
Published: January 2020

Vaccines which successfully protect against virus infections usually need to induce a broadly reactive immune response which includes the induction of cytotoxic T lymphocytes (CTL). In this study, we have used a convenient in vitro approach to investigate if plasmid DNAs encoding proteins of herpes simplex virus (HSV) are capable of inducing primary CD8+ CTL. Dendritic cells or macrophages were transfected with either plasmid DNA encoding glycoprotein B or DNA encoding the immediate-early protein ICP27. These antigen-presenting cells (APC) were then used to stimulate enriched populations of naive T cells in microcultures for 5 days in vitro. Antigen-specific CD8+ CTL which reacted both with specific protein-expressing targets and with syngeneic targets infected with HSV could be demonstrated. Dendritic cells, as APC, generated the maximal responses, but such cells needed to be transfected with DNA in the presence of a cationic lipid. However, macrophages could act as APC when they were exposed to purified DNA. HSV-primed splenocytes were also shown to generate specific CTL responses when they were stimulated with purified DNA encoding ICP27. The novel approach described in this paper promises to be extremely useful, since defining immunogenicity profiles and identifying epitopes on viral proteins should be easier and more convenient when working with DNA and investigating variables in vitro. This is particularly the case with complex viruses such as HSV, most of whose encoded proteins have yet to be isolated in sufficient quantity or purity to perform in vivo immunological studies.
Murine AIDS is an antigen-driven disease: requirements for major histocompatibility complex class II expression and CD4+ T cells
Authors: Giese N A , Giese T , Morse H C
Published: January 2020

Murine AIDS (MAIDS) is a complex syndrome of lymphoproliferation and immunodeficiency induced by a replication-defective murine leukemia virus (BM5def) that encodes Pr60gag as its only product. It has been suggested that the gag polyprotein is responsible for vigorous antigenic stimulation of CD4+ T cells and generalized secondary activation of the immune system. This model was tested first by infecting mice (C2K/O) that lack class II major histocompatibility complex molecules required for presentation of antigens to CD4+ T cells. C2K/O mice expressed BM5def at high levels but did not develop MAIDS either when unmanipulated or following transfer of CD4+ T cells. Second, B6 mice reconstituted with C2K/O bone marrow cells had normal frequencies of B cells (class II negative) and CD4+ cells and expressed high levels of BM5def transcripts but did not develop MAIDS; however, MAIDS developed in class II-competent nu/nu mice reconstituted with CD4+ T cells and in C2K/O mice reconstituted with B6 bone marrow to give class II-positive B cells and with purified CD4+ T cells. These results indicate that induction of MAIDS by BM5def is antigen driven and is dependent on expression of major histocompatibility complex class II molecules on antigen-presenting cells and the presence of CD4+ T cells.
DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein
Authors: Tsuchihashi Z , Brown P O
Published: January 2020

Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified. The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations. We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules. Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing). NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation. This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications.
The human poliovirus receptor alpha is a serine phosphoprotein
Authors: Bibb J A , Bernhardt G , Wimmer E
Published: January 2020

The human receptors for poliovirus (hPVR) are members of the immunoglobulin superfamily. Whereas the two membrane-bound isoforms, hPVR alpha and hPVR delta, share identical three-domain extracellular portions, their C-terminal cytoplasmic parts differ considerably. This feature is well conserved in the corresponding monkey proteins AGM alpha 1, AGM delta 1, and AGM alpha 2. The cellular function of these proteins is presently unknown. In this short communication we report that hPVR alpha and possibly also AGM alpha 1 and AGM alpha 2, but not the delta isoforms, are phosphoproteins. The phosphorylation occurs at a serine in the cytoplasmic tails of these receptors. We further present evidence suggesting that the kinase responsible for the phosphorylation is calcium/calmodulin kinase II.
Mutation of the bovine papillomavirus E5 oncoprotein at amino acid 17 generates both high- and low-transforming variants
Authors: Sparkowski J , Anders J , Schlegel R
Published: January 2020

The E5 transforming protein of bovine papillomavirus type 1 is a 44-amino-acid, hydrophobic protein which localizes predominantly to Golgi membranes. The E5 transmembrane domain contains a highly conserved glutamine residue at position 17 which, from previous limited mutagenic analysis, appeared essential for transforming activity. In order to determine the specific amino acid requirements at this position, we constructed a series of substitution mutants, representing all classes of amino acids, employing a vector which expressed E5 independently of other bovine papillomavirus gene products. All of the expressed E5 mutant proteins were stable, dimerized normally, and localized to the Golgi. Our results obtained with C127 mouse cells demonstrated that acidic amino acids (and serine) increased E5 transforming activity, whereas basic amino acids greatly inhibited E5 activity. Nonpolar amino acid substitutions were also defective. Interestingly, the relative transforming activities of these E5 mutant proteins changed dramatically when assayed with NIH 3T3 cells, suggesting that an auxiliary cellular protein(s) may modulate E5 transformation or that there are additional or different mechanisms of E5 transformation which are utilized in these two cell lines.
Efficient system of homologous RNA recombination in brome mosaic virus: sequence and structure requirements and accuracy of crossovers
Authors: Nagy P D , Bujarski J J
Published: January 2020

Brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants engineered to support intersegment RNA recombination, was used for the determination of sequence and structural requirements of homologous crossovers. A 60-nucleotide (nt) sequence, common between wild-type RNA2 and mutant RNA3, supported efficient repair (90%) of a modified 3' noncoding region in the RNA3 segment by homologous recombination with wild-type RNA2 3' noncoding sequences. Deletions within this sequence in RNA3 demonstrated that a nucleotide identity as short as 15 nt can support efficient homologous recombination events, while shorter (5-nt) sequence identity resulted in reduced recombination frequency (5%) within this region. Three or more mismatches within a downstream portion of the common 60-nt RNA3 sequence affected both the incidence of recombination and the distribution of crossover sites, suggesting that besides the length, the extent of sequence identity between two recombining BMV RNAs is an important factor in homologous recombination. Site-directed mutagenesis of the common sequence in RNA3 did not reveal a clear correlation between the stability of predicted secondary structures and recombination activity. This indicates that homologous recombination does not require similar secondary structures between two recombining RNAs at the sites of crossovers. Nearly 20% of homologous recombinants were imprecise (aberrant), containing either nucleotide mismatches, small deletions, or small insertions within the region of crossovers. This implies that homologous RNA recombination is not as accurate as proposed previously. Our results provide experimental evidence that the requirements and thus the mechanism of homologous recombination in BMV differ from those of previously described heteroduplex-mediated nonhomologous recombination (P. D. Nagy and J. J. Bujarski, Proc. Natl. Acad. Sci. USA 90:6390-6394, 1993).
The effect of two closely inserted transcription consensus sequences on coronavirus transcription
Authors: Joo M , Makino S
Published: January 2020

Insertion of an intergenic region from the murine coronavirus mouse hepatitis virus into a mouse hepatitis virus defective interfering (DI) RNA led to transcription of subgenomic DI RNA in helper virus-infected cells. Using this system, we studied how two intergenic regions in close proximity affected subgenomic RNA synthesis. When two intergenic regions were separated by more than 100 nucleotides, slightly less of the larger subgenomic DI RNA (synthesized from the upstream intergenic region) was made; this difference was significant when the intergenic region separation was less than about 35 nucleotides. Deletion of sequences flanking the two intergenic regions inserted in close proximity did not affect transcription. No significant change in the ratio of the two subgenomic DI RNAs was observed when the sequence between the two intergenic regions was altered. Removal of the downstream intergenic region restored transcription of the larger subgenomic DI RNA. The UCUAAAC consensus sequence was needed for efficient suppression of the larger subgenomic DI RNA synthesis. These results demonstrated that the downstream intergenic sequence was suppressing subgenomic DI RNA synthesis from the upstream intergenic region. We discuss possible mechanisms to account for the regulation of this suppression of subgenomic DI RNA synthesis and the ways in which they relate to the general regulation of coronavirus transcription.
Human immunodeficiency virus type 1 MA deletion mutants expressed in baculovirus-infected cells: cis and trans effects on the Gag precursor assembly pathway
Authors: Chazal N , Gay B , Carrière C , Tournier J , Boulanger P
Published: January 2020

The role of the matrix protein (MA) of human immunodeficiency virus type 1 in intracellular transport, assembly, and extracellular release of Gag polyprotein precursor (Pr55gag) was investigated by deletion mutagenesis of the MA domain of recombinant Gag precursor expressed in baculovirus-infected cells. In addition, three carboxy-terminally truncated forms of the Gag precursor, representing mainly the MA, were constructed. One corresponded to an MA with a deletion of its last 12 residues (amb120), while the others corresponded to the entire MA with an additional sequence from the N-terminal portion of the CA (amb143 and och180). Deletions within the MA central region (residues 41 to 78) appeared to be detrimental to Gag particle assembly and budding from the plasma membrane. A slightly narrower domain, between amino acids 41 and 68, was found to be critical for soluble Gag secretion. Mutations which totally or partially deleted one or the other of the two polybasic signals altered the transport of N-myristylated Gag precursor to the plasma membrane. In coexpression with wild-type Gag precursor, a discrete trans-dominant negative effect on wild-type Gag particle assembly and release was observed with deletion mutants located in the central MA region (residues 41 to 78). A more significant negative effect was obtained with the two recombinant proteins of amb120 and och180, which redirected the Gag particle assembly pathway from the plasma membrane compartment to intracellular vesicles (amb120) and to the nuclear compartment (och180).
Long terminal repeat enhancer core sequences in proviruses adjacent to c-myc in T-cell lymphomas induced by a murine retrovirus
Authors: Morrison H L , Soni B , Lenz J
Published: January 2020

The transcriptional enhancer in the long terminal repeat (LTR) of the T-lymphomagenic retrovirus SL3-3 differs from that of the nonleukemogenic virus Akv at several sites, including a single base pair difference in an element termed the enhancer core. Mutation of this T-A base pair to the C-G C-G sequence found in Akv significantly attenuated the leukemogenicity of SL3-3. Thus, this difference is important for viral leukemogenicity. Since Akv is an endogenous virus, this suggests that the C-G in its core is an adaptation to being minimally pathogenic. Most tumors that occurred in mice inoculated with the mutant virus, called SAA, contained proviruses with reversion or potential suppressor mutations in the enhancer core. We also found that the 72-bp tandem repeats constituting the viral enhancer could vary in number. Most tumors contained mixtures of proviruses with various numbers of 72-bp units, usually between one and four. Variation in repeat number was most likely due to recombination events involving template misalignment during viral replication. Thus, two processes during viral replication, misincorporation and recombination, combined to alter LTR enhancer structure and generate more pathogenic variants from the mutant virus. In SAA-induced tumors, enhancers of proviruses adjacent to c-myc had the largest number of core reversion or suppressor mutations of all of the viral enhancers in those tumors. This observation was consistent with the hypothesis that one function of the LTR enhancers in leukemogenesis is to activate proto-oncogenes such as c-myc.
Congo red prolongs the incubation period in scrapie-infected hamsters
Authors: Ingrosso L , Ladogana A , Pocchiari M
Published: January 2020

In scrapie-infected cells, Congo red inhibits both the replication of the infectious agent and accumulation of the protease-resistant form of PrP (PrP-res). In this report, we show that Congo red prolongs the incubation periods of hamsters experimentally infected with two different strains of scrapie.
Mouse hepatitis virus receptor activities of an MHVR/mph chimera and MHVR mutants lacking N-linked glycosylation of the N-terminal domain
Authors: Dveksler G S , Basile A A , Cardellichio C B , Holmes K V
Published: January 2020

Mouse hepatitis virus binds to the N-terminal domain of its receptor, MHVR, a murine biliary glycoprotein with four immunoglobulin-like domains (G.S. Dveksler, M. N. Pensiero, C. W. Dieffenbach, C. B. Cardellichio, A.A. Basile, P.E. Elia, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 90:1716-1720, 1993). A recombinant protein with only the anchored N-terminal domain was not a functional receptor, but a recombinant protein with the N-terminal domain of MHVR linked to the second and third immunoglobulin-like domains and anchor from the mouse poliovirus receptor homolog, mph, was a functional receptor for mouse hepatitis virus. The native four-domain MHVR has 16 potential N-linked glycosylation sites, including three on the N-terminal domain. Recombinant proteins lacking each one of these three sites or all three of them were functional receptors. Thus, glycosylation of the N-terminal domain is not required, but a glycoprotein longer than the N-terminal domain is required for virus receptor activity.
Transcription factor Oct1 binds to the AT-rich segment of the simian virus 40 replication origin
Authors: Kilwinski J , Baack M , Heiland S , Knippers R
Published: January 2020

A cellular protein that binds to the AT-rich late segment of the simian virus 40 (SV40) origin of replication has been identified as transcription factor Oct1. This conclusion is based on the following observations: the late origin binding protein has a molecular mass of about 100 kDa, like factor Oct1, and shares other biochemical properties with Oct1; its binding to the origin is inhibited by antibodies directed against the POU domain of factor Oct1; the isolated POU domain of Oct1 specifically binds to the SV40 late origin region. Thus, the SV40 genome contains binding sites for transcription factor Oct1 in the origin of replication in addition to the previously characterized octamer sites in the viral promoter enhancer. Oct1, bound to the viral origin, negatively affects the DNA unwinding reaction catalyzed by the viral replication initiator T antigen, suggesting that Oct1 may have a role in the regulation of viral replication.
Mutational analysis of the leucine zipper motif in the Newcastle disease virus fusion protein
Authors: Reitter J N , Sergel T , Morrison T G
Published: January 2020

The paramyxovirus fusion proteins have a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit (R. Buckland and F. Wild, Nature [London] 338:547, 1989). To determine the role of the conserved leucines in the oligomeric structure and biological activity of the Newcastle disease virus (NDV) fusion protein, the heptadic leucines at amino acids 481, 488, and 495 were changed individually and in combination to an alanine residue. While single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein, although cell surface expression of the mutants was higher than that of the wild-type protein. Substitution of all three leucine residues with alanine did not alter the size of the fusion protein oligomer as determined by sedimentation in sucrose gradients. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain, resulted in secretion of an oligomeric polypeptide. These results indicate that the conserved leucines are not necessary for oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative changes of serine to alanine (position 473), glutamic acid to lysine or alanine (position 482), asparagine to lysine (position 485), or aspartic acid to alanine (position 489), the fusogenic ability of the protein was not significantly disrupted. In addition, a double mutant (E482A,D489A) which removed negative charges along one side of the helix had negligible effects on fusion activity.
Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol precursor incorporation
Authors: Srinivasakumar N , Hammarskjöld M L , Rekosh D
Published: January 2020

The core of human immunodeficiency virus type 1 is derived from two precursor polyproteins, Pr55gag and Pr160gag-pol. The Gag precursor can assemble into immature virus-like particles when expressed by itself, while the Gag-Pol precursor lacks particle-forming ability. We have shown previously that the Gag precursor is able to "rescue" the Gag-Pol precursor into virus-like particles when the two polyproteins are expressed in the same cell by using separate simian virus 40-based plasmid expression vectors. To understand this interaction in greater detail, we have made deletion mutations in the capsid-coding regions of Gag- and Gag-Pol-expressing plasmids and assayed for the abilities of these precursors to assemble into virus-like particles. When we tested the abilities of Gag-Pol precursors to be incorporated into particles of Gag by coexpressing the precursors, we found that mutant Gag-Pol precursors lacking a conserved region in retroviral capsid proteins, the major homology region (MHR), were excluded from wild-type Gag particles. Mutant precursors lacking MHR were also less efficient in processing the Gag precursor in trans. These results suggest that the MHR is critical for interactions between Gag and Gag-Pol molecules. In contrast to these results, expression of mutated Gag precursors alone showed that deletions in the capsid region, including those which removed the MHR, reduced the efficiency of particle formation by only 40 to 50%. The mutant particles, however, were clearly lighter than the wild type in sucrose density gradients. These results indicate that the requirements for Gag particle formation differ from the ones essential for efficient incorporation of the Gag-Pol precursor into these particles.
Virus-specific antigen presentation by different subsets of cells from lung and mediastinal lymph node tissues of influenza virus-infected mice
Authors: Hamilton-Easton A , Eichelberger M
Published: January 2020

Immune responses at mucosal sites are thought to be initiated in the draining lymph nodes, where dendritic cells present viral antigens and induce naive T cells to proliferate and to become effectors. Formal proof that antigen-presenting cells (APC) do indeed localize to the regional lymph nodes has been lacking for viral infections of the respiratory tract. Influenza virus was detected in the draining mediastinal lymph nodes (MLN) early after intranasal inoculation, with peak virus titers in this tissue measured at 2 days postinfection. Virus-specific cytotoxic T-lymphocyte responses were first detected in the MLN 1 day later. Macrophages, dendritic cells, and B lymphocytes were isolated from influenza virus-infected mice and assayed for the capacity to stimulate a major histocompatibility complex class I-restricted virus-specific T-cell hybridoma. All APC populations from lungs and MLN contained virus and thus had the potential to present antigen to CD8+ T cells. The APC recovered from the lungs of influenza virus-infected mice and dendritic cells from the MLN were able to stimulate virus-specific responses. The lack of a virus-specific T-cell response to B cells corresponds to the small number of virus-positive B lymphocytes in the MLN. These results indicate that dendritic cells and macrophages are antigen positive in mice acutely infected with an influenza A virus and that dendritic cells are probably responsible for initiating the cytotoxic T-lymphocyte response to influenza virus in the draining lymph nodes.
Differential proteolytic processing leads to multiple forms of the CA protein in avian sarcoma and leukemia viruses
Authors: Pepinsky R B , Papayannopoulos I A , Chow E P , Krishna N K , Craven R C , Vogt V M
Published: January 2020

The CA (capsid) protein of avian sarcoma and leukemia viruses occurs in multiple species. Only one form has been previously characterized biochemically. We have now determined that the mature CA protein of avian sarcoma and leukemia viruses exists as three species with different C termini, ending in amino acid residues A-476, A-478, and M-479 of the Gag precursor, respectively. These structures were deduced from a combination of cyanogen bromide peptide mapping, sequence analysis of tryptic peptides, and electrospray mass spectrometry. The three forms of CA were detected in the same ratios in Rous sarcoma virus and avian myeloblastosis virus and therefore are likely to represent a common feature of members of this genus of avian retroviruses. The only previously reported CA species, CAM-479, accounts for only about 36% of the total CA protein, while CAA-476 and CAA-478 account for 55 and 9%, respectively. From the analysis of peptides cleaved in vitro by PR, the viral protease, we infer that the cleavage site between A-476 and A-477 not only is recognized by PR but is the preferred site. We were unable to determine if A-478/A-479 is a cleavage site for PR or alternatively if CAA-478 results from further processing of CAM-479 by a carboxypeptidase. To study the biological significance of residues A-477 to M-479, we constructed genetically altered viruses in which deletions removed either residues 477 to 479 or 477 to 488. The resulting virus particles appeared to assembly with normal efficiencies, but the latter mutant showed slowed proteolytic processing. Neither of the mutants was infectious.
Initial characterization of the membrane-associated form of ICP4 of herpes simplex virus type 1
Authors: Xia L , Courtney R J
Published: January 2020

The immediate-early gene product, ICP4, of herpes simplex virus type 1 (HSV-1) is one of the major transcriptional regulatory proteins in the virus replicative process and is localized primarily within the nucleus soon after its synthesis. Earlier studies have shown that detectable amounts of ICP4 are also associated with the plasma membranes of infected cells (F. Yao and R. J. Courtney, J. Virol. 65:1516-1524, 1991). To extend our understanding of the properties of the membrane-associated ICP4, we have used various electrophoretic techniques, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis, and isoelectric focusing, to compare the membrane- and nuclear-associated forms of ICP4. The data from all of these methods revealed that a single unique form of ICP4 associates with plasma membranes of HSV-1-infected cells. While multiple forms of ICP4 were detected in infected cell nuclei, the membrane-associated form of ICP4 appeared to have a lower apparent molecular weight and a more acidic pI than the various forms of ICP4 found in infected cell nuclei. These results suggest that a novel form of ICP4 may associate with plasma membranes of HSV-1-infected cells. A recombinant adenovirus, AdICP4 (encoding an ICP4 protein), was used to determine the role that other herpesvirus proteins may play in the membrane association of ICP4. The results suggest that the expression of other HSV-1 proteins is not required for the membrane association of ICP4.
Interactions of the transcription factor AP-1 with the long terminal repeat of different human immunodeficiency virus type 1 strains in Jurkat, glial, and neuronal cells
Authors: Canonne-Hergaux F , Aunis D , Schaeffer E
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) infection of the neuronal and astroglial cells of the central nervous system has been proposed to contribute to HIV-1-associated dementia. Recently it was shown that differences in the nucleotide sequence of the long terminal repeat (LTR) of different HIV-1 strains govern the tissue-specific pattern of viral expression. The LTR from central nervous system-derived HIV-1 strains JR-FL and JR-CSF directs expression in the neurons of transgenic mice, in contrast with the lymphotropic LAI strain. By in vitro footprinting, gel retardation, and methylation interference experiments, we have studied the interactions of host cell proteins from human neuronal, glial, HeLa, and Jurkat T cells with the LTRs from the neurotropic JR-FL and JR-CSF strains, compared with the LAI strain. Proteins belonging to the nuclear receptor family bind with different affinities to variant -352 to -324 sites. Gel supershift assays with Jun and Fos antibodies showed that the AP-1 transcription factor present in the various cell types was unable to recognize the -352 to -324 and -306 to -285 AP-1 putative binding sites. Interestingly, Jun and Fos components of AP-1 interact with the variant TGGCTCA sequence located in the -247 to -222 region of both neurotropic strains. These interactions were cell type specific, since they were detected only with extracts from glial and HeLa cells and not from neuronal or Jurkat cells. Cotransfection experiments further revealed that the -247 to -222 sequence is able to mediate AP-1-induced transcriptional activation in glial and not neuronal cells.
A leucine triplet repeat sequence (LXX)4 in p6gag is important for Vpr incorporation into human immunodeficiency virus type 1 particles
Authors: Lu Y L , Bennett R P , Wills J W , Gorelick R , Ratner L
Published: January 2020

Incorporation of Vpr into human immunodeficiency virus type 1 (HIV-1) virions is mediated by the Gag protein, independently of other viral components. We have coexpressed Vpr and Gag constructs in a vaccinia virus expression system in order to map the region of Gag involved in Vpr packaging. Deletion of the carboxyl-terminal p6 region of Gag impaired the ability of Gag to package Vpr. To confirm the role of p6 in Vpr packaging, Rous sarcoma virus (RSV)-HIV chimeras containing HIV-1 p6 were constructed. Although RSV Gag does not package Vpr into virus particles, a chimera containing HIV-1 p6 is sufficient for Vpr incorporation. To map the region of p6 involved in Vpr packaging, a series of p6 point mutations and deletion mutations was analyzed. Mutations in the N-terminal p6 proline-rich domain, for which preliminary evidence shows a marked decrease in virion incorporated RNA, did not affect Vpr incorporation. Deletion of residues 1 to 31 of HIV-1 p6 did not affect Vpr packaging, but residues 35 to 47, including an (LXX)4 domain, were required for Vpr incorporation into virus particles.