bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of domains within the human cytomegalovirus major immediate-early 86-kilodalton protein and the retinoblastoma protein required for physical and functional interaction with each other
Authors: Fortunato E A , Sommer M H , Yoder K , Spector D H
Published: January 2020

The human cytomegalovirus major immediate-early 86-kDa protein (IE2 86) plays an important role in the trans activation and regulation of HCMV gene expression. Previously, we demonstrated that IE2 86 contains three regions (amino acids [aa] 86 to 135, 136 to 290, and 291 to 364) that can independently bind to in vitro-translated Rb when IE2 86 is produced as a glutathione S-transferase fusion protein (M. H. Sommer, A. L. Scully, and D. H. Spector, J. Virol. 68:6223-6231, 1994). In this report, we have elucidated the regions of Rb involved in binding to IE2 86 and have further analyzed the functional nature of the interaction between these two proteins. We find that two domains on Rb, the A/B pocket and the carboxy terminus, can each independently form a complex with IE2 86. In functional assays, we demonstrate that IE2 86 and another IE protein, IE1 72, can counter the enlarged flat cell phenotype, but not the G1/S block, which results from expression of wild-type Rb in the human osteosarcoma cell line Saos-2. Mutational analysis reveals that there are two domains on IE2 86 that can independently affect Rb function. One region (aa 241 to 369) includes the major Rb-binding domain, while the second maps to the amino-terminal region (aa 1 to 85) common to both IE2 86 and IE1 72. These data show that Rb and IE2 86 physically and functionally interact with each other via at least two separate domains and provide further support for the hypothesis that IE2 86 may exert its pleiotropic effects through the formation of multimeric protein complexes.
Neutralizing antibodies against the V3 loop of human immunodeficiency virus type 1 gp120 block the CD4-dependent and -independent binding of virus to cells
Authors: Valenzuela A , Blanco J , Krust B , Franco R , Hovanessian A G
Published: January 2020

The CD4 molecule is an essential receptor for human immunodeficiency virus type 1 (HIV-1) through high-affinity interactions with the viral external envelope glycoprotein gp120. Previously, neutralizing monoclonal antibodies (MAbs) specific to the third hypervariable domain of gp120 (the V3 loop) have been thought to block HIV infection without affecting the binding of HIV particles to CD4-expressing human cells. However, here we demonstrate that this conclusion was not correct and was due to the use of soluble gp120 instead of HIV particles. Indeed, neutralizing anti-V3 loop MAbs inhibited completely the binding and entry of HIV particles into CD4+ human cells. In contrast, the binding of virus was only partially inhibited by neutralizing anti-CD4 MAbs against the gp120 binding site in CD4, which, like the anti-V3 loop MAbs, completely inhibited HIV entry and infection. Nonneutralizing control MAbs against either the V3 loop or the N or C terminus of gp120 had no significant effect on HIV binding and entry. HIV-1 particles were also found to bind human and murine cells expressing or not expressing the human CD4 molecule. Interestingly, the binding of HIV to CD4+ murine cells was inhibited by both anti-V3 and anti-CD4 MAbs, whereas the binding to human and murine CD4- cells was affected only by anti-V3 loop MAbs. The effect of anti-V3 loop neutralizing MAbs on the HIV binding to cells appears not to be the direct consequence of gp120 shedding from HIV particles or of a decreased affinity of CD4 or gp120 for binding to its surface counterpart. Taken together, our results suggest the existence of CD4-dependent and -independent binding events involved in the attachment of HIV particles to cells; in both of these events, the V3 loop plays a critical role. As murine cells lack the specific cofactor CXCR4 for HIV-1 entry, other cell surface molecules besides CD4 might be implicated in stable binding of HIV particles to cells.
The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis
Authors: Shisler J , Yang C , Walter B , Ware C F , Gooding L R
Published: January 2020

Cytotoxic T cells use Fas (CD95), a member of the tumor necrosis factor (TNF) receptor superfamily, to eliminate virus-infected cells by activation of the apoptotic pathway for cell death. The adenovirus E3 region encodes several proteins that modify immune defenses, including TNF-dependent cell death, which may allow this virus to establish a persistent infection. Here we show that, as an early event during infection, the adenovirus E3-10.4K/14.5K complex selectively induces loss of Fas surface expression and blocks Fas-induced apoptosis of virus-infected cells. Loss of surface Fas occurs within the first 4 h postinfection and is not due to decreased production of Fas protein. The decrease in surface Fas is distinct from the 10.4K/14.5K-mediated loss of the epidermal growth factor receptor on the same cells, because intracellular stores of Fas are not affected. Further, 10.4K/14.5K, which was previously shown to protect against TNF cytolysis, does not induce a loss of TNF receptor, indicating that this complex mediates more than one function to block host defense mechanisms. These results suggest yet another mechanism by which adenovirus modulates host cytotoxic responses that may contribute to persistent infection by human adenoviruses.
In vitro polymerase activity of Thogoto virus: evidence for a unique cap-snatching mechanism in a tick-borne orthomyxovirus
Authors: Leahy M B , Dessens J T , Nuttall P A
Published: January 2020

The tick-borne Thogoto virus (THOV) is the type species of a new genus in the family Orthomyxoviridae. Its genome comprises six segments of single-stranded, negative-sense RNA. Each segment possesses conserved regions of semicomplementary nucleotides at the 3' and 5' termini which strongly resemble those of influenza virus. An in vitro polymerase assay based on reconstituted THOV viral cores was developed, and activity was shown to rely on an interaction between the conserved 3'- and 5'-terminal sequences and to be primer dependent. Addition of globin mRNA primed transcription, catalyzing the addition of an extra nucleotide to the transcripts, corresponding to the 5'-terminal m7G cap residue. Priming with various cap analogs suggested that THOV transcription is initiated preferentially with m7GpppAm and involves base pairing. This is the first experimental evidence of endonuclease activity in THOV as part of a unique cap-snatching mechanism.
Receptor-targeted recombinant adenovirus conglomerates: a novel molecular conjugate vector with improved expression characteristics
Authors: Schwarzenberger P , Hunt J D , Robert E , Theodossiou C , Kolls J K
Published: January 2020

To develop improved strategies for gene transfer to hematopoietic cells, we have explored targeted gene transfer using molecular conjugate vectors (MCVs). MCVs are constructed by condensing plasmid DNA containing the gene of interest with polylysine (PL), PL linked to a replication-incompetent adenovirus (endosomolytic agent), and PL linked to streptavidin for targeting with biotinylated ligands. In this report, we compare gene transfer to K562 cells by using the previously described transferrin-targeted MCV (Trans-MCV) to a novel transferrin-targeted MCV. In the novel MCV, the transferred gene (luciferase) is in the genome of recombinant replication-incompetent adenovirus (recMCV), which also acts as the endosomolytic agent. The level of luciferase gene expression was fivefold higher in K562 cells transfected with Trans-recMCV than in cells transfected with Trans-MCV. Furthermore, targeted transfection with recMCV resulted in prolonged luciferase expression that declined 14 to 20 days after transfection, in comparison with Trans-MCV, where luciferase expression declined by 4 to 8 days. Moreover, targeted transfection of K562 cells with the Trans-recMCV resulted in persistent luciferase gene expression for 6 months. Analysis of luciferase gene expression in K562 single-cell clones that were subcloned 5 weeks after transfection with Trans-recMCV showed that 35 to 50% of the single-cell clones had intermediate to high levels of luciferase gene expression that was stable for 6 months, with the remaining clones showing low or no luciferase gene expression. Stable gene expression was associated with integration of adenovirus sequences into genomic DNA.
Rotavirus virus-like particles administered mucosally induce protective immunity
Authors: O'Neal C M , Crawford S E , Estes M K , Conner M E
Published: January 2020

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.
Utilization of chemokine receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses
Authors: Rucker J , Edinger A L , Sharron M , Samson M , Lee B , Berson J F , Yi Y , Margulies B , Collman R G , Doranz B J , Parmentier M , Doms R W
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.
Identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants
Authors: Saeki K , Ohtsuka N , Taguchi F
Published: January 2020

We previously demonstrated by site-directed mutagenesis analysis that the amino acid residues at positions 62 and 214 to 216 in the N-terminal region of mouse hepatitis virus (MHV) spike (S) protein are important for receptor-binding activity (H. Suzuki and F. Taguchi, J. Virol. 70:2632-2636, 1996). To further identify the residues responsible for the activity, we isolated the mutant viruses that were not neutralized with the soluble form of MHV receptor proteins, since such mutants were expected to have mutations in amino acids responsible for receptor-binding activity. Five soluble-receptor-resistant (srr) mutants isolated had mutations in a single amino acid at three different positions: one was at position 65 (Leu to His) (srr11) in the S1 subunit and three were at position 1114 (Leu to Phe) (srr3, srr4, and srr7) and one was at position 1163 (Cys to Phe) (srr18) in the S2 subunit. The receptor-binding activity examined by a virus overlay protein blot assay and by a coimmunoprecipitation assay showed that srr11 S protein had extremely reduced binding activity, while the srr7 and srr18 proteins had binding activity similar to that of wild-type cl-2 protein. However, when cell surface receptors were used for the binding assay, all srr mutants showed activity similar to that of the wild type or only slightly reduced activity. These results, together with our previous observations, suggest that amino acids located at positions 62 to 65 of S1, a region conserved among the MHV strains examined, are important for receptor-binding activity. We also discuss the mechanism by which srr mutants with a mutation in S2 showed high resistance to neutralization by a soluble receptor, despite their sufficient level of binding to soluble receptors.
A mutation in tomato aspermy cucumovirus that abolishes cell-to-cell movement is maintained to high levels in the viral RNA population by complementation
Authors: Moreno I M , Malpica J M , Rodríguez-Cerezo E , García-Arenal F
Published: January 2020

The nucleotide substitution C-->A at nucleotide 100 of tomato aspermy cucumovirus (TAV) strain V (V-TAV) RNA segment 3 (RNA3) introduces an ocher stop at the fourth codon of the movement protein open reading frame. Experiments with RNA transcripts from full-length clones showed that this mutation abolished cell-to-cell movement and, thus, infectivity in planta. Heterogeneity analyses on stock V-TAV virion RNA showed that an A at position 100 was present in the molecular population of RNA3 at a frequency of 0.76 and that a C at this position was present at a frequency of 0.24. This result indicates that a fraction of RNA3 molecules complements cell-to-cell movement of movement-defective molecules. It was shown that the mutation C-->A conferred enhanced RNA replication of the defective mutant in tobacco protoplasts. The effect of the mutation on replication was dependent on sequence context, since the same mutation did not affect the replication efficiency in the related TAV strain 1 RNA3. Competition experiments in tobacco protoplasts were done to estimate the fitness during a cell invasion cycle of the movement-defective mutant relative to the wild type (wt). From these data, a lower limit to the degree of complementation of movement-defective molecules by movement-competent ones could be estimated as 0.13. This estimate shows that complementation may play an important role in the determination of genetic structure in RNA genome populations. A further effect of the enhanced replication of the movement-defective mutant was the efficient competition with the wt for the initiation of infection foci in planta.
Distinct functions and requirements for the Cys-His boxes of the human immunodeficiency virus type 1 nucleocapsid protein during RNA encapsidation and replication
Authors: Schwartz M D , Fiore D , Panganiban A T
Published: January 2020

The process of retroviral RNA encapsidation involves interaction between trans-acting viral proteins and cis-acting RNA elements. The encapsidation signal on human immunodeficiency virus type 1 (HIV-1) RNA is a multipartite structure composed of functional stem-loop structures. The nucleocapsid (NC) domain of the Gag polyprotein precursor contains two copies of a Cys-His box motif that have been demonstrated to be important in RNA encapsidation. To further characterize the role of the Cys-His boxes of the HIV-1 NC protein in RNA encapsidation, the relative efficiency of RNA encapsidation for virus particles that contained mutations within the Cys-His boxes was measured. Mutations that disrupted the first Cys-His box of the NC protein resulted in virus particles that encapsidated genomic RNA less efficiently and subgenomic RNA more efficiently than did wild-type virus. Mutations within the second Cys-His box did not significantly affect RNA encapsidation. In addition, a full complement of wild-type NC protein in virus particles is not required for efficient RNA encapsidation or virus replication. Finally, both Cys-His boxes of the NC protein play additional roles in virus replication.
The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function
Authors: Sheng Q , Denis D , Ratnofsky M , Roberts T M , DeCaprio J A , Schaffhausen B
Published: January 2020

Tumor suppressors of the retinoblastoma susceptibility gene family regulate cell growth and differentiation. Polyomavirus large T antigens (large T) bind Rb family members and block their function. Mutations of large T sequences conserved with the DnaJ family affect large T binding to a cellular DnaK, heat shock protein 70. The same mutations abolish large T activation of E2F-containing promoters and Rb binding-dependent large T activation of cell cycle progression. Cotransfection of a cellular DnaJ domain blocks wild-type large T action, showing that the connection between the chaperone system and tumor suppressors is direct. Although they are inactive in assays dependent on Rb family binding, mutants in the J region retain the ability to associate with pRb, p107, and p130. This suggests that binding of Rb family members by large T is not sufficient for their inactivation and that a functional J domain is required as well. This work connects the DnaJ and DnaK molecular chaperones to regulation of tumor suppressors by polyomavirus large T.
Rotavirus nonstructural glycoprotein NSP4 alters plasma membrane permeability in mammalian cells
Authors: Newton K , Meyer J C , Bellamy A R , Taylor J A
Published: January 2020

The endoplasmic reticulum-localized transmembrane glycoprotein NSP4 of rotavirus is a key protein involved in rotavirus cytopathology. We have used a dual-recombinant vaccinia virus system to express NSP4 in monkey kidney epithelial cells at a level comparable to that observed during rotavirus infection. Expression of NSP4 results in loss of plasma membrane integrity, which can be demonstrated by release of both 51Cr and lactate dehydrogenase into the medium. The cytotoxic behavior of NSP4 is dose dependent, and morphological analysis reveals gross changes to cell ultrastructure, indicative of cell death. Thus, intracellular expression of a single rotavirus protein which localizes to the endoplasmic reticulum membrane has profound effects on the stability of the plasma membrane and cell viability. Analysis of NSP4 deletion mutants indicates that a membrane-proximal region located within the cytoplasmic domain may mediate cytotoxicity.
The 3' untranslated region of the B19 parvovirus capsid protein mRNAs inhibits its own mRNA translation in nonpermissive cells
Authors: Pallier C , Greco A , Le Junter J , Saib A , Vassias I , Morinet F
Published: January 2020

Although parvoviruses are found throughout the animal kingdom, only the human pathogenic B19 virus has so far been shown to possess a limited host range, with erythroid progenitor cells as the main target cells supporting B19 propagation. The underlying mechanism of such erythroid tropism is still unexplained. Synthesis of the NS1 nonstructural protein occurs in permissive and nonpermissive cells, such as megakaryocytes, whereas synthesis of the VP1 and VP2 capsid proteins seems to be restricted to burst-forming units and CFU of erythroid cells. In nonpermissive cells, the NS1 protein is overexpressed and the NS1 RNAs are the predominant RNA species. However, the VP1 and VP2 proteins are not detectable, although the corresponding mRNAs are synthesized. Since all transcripts have part of the 5' untranslated region (5' UTR) in common but distinct 3' UTRs characterizing the nonstructural- and structural-protein mRNAs, we investigated, in transient transfection assays, the possible involvement of the 3' UTR of the capsid protein mRNAs in VP1 and VP2 protein synthesis in nonpermissive Cos cells. The results showed that (i) the 3' UTR of mRNAs coding for the capsid proteins repressed VP1 and VP2 protein synthesis, (ii) the 3' UTR did not affect nuclear export or mRNA stability, and (iii) mRNAs bearing the 3' UTR of the capsid protein mRNAs did not associate with ribosomes at all. Taken together, these results indicate that in nonpermissive cells, the 3' UTR of the capsid protein mRNAs represses capsid protein synthesis at the translational level by inhibiting ribosome loading.
The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range
Authors: Schickli J H , Zelus B D , Wentworth D E , Sawicki S G , Holmes K V
Published: January 2020

In murine 17 Cl 1 cells persistently infected with murine coronavirus mouse hepatitis virus strain A59 (MHV-A59), expression of the virus receptor glycoprotein MHVR was markedly reduced (S. G. Sawicki, J. H. Lu, and K. V. Holmes, J. Virol. 69:5535-5543, 1995). Virus isolated from passage 600 of the persistently infected cells made smaller plaques on 17 Cl 1 cells than did MHV-A59. Unlike the parental MHV-A59, this variant virus also infected the BHK-21 (BHK) line of hamster cells. Virus plaque purified on BHK cells (MHV/BHK) grew more slowly in murine cells than did MHV-A59, and the rate of viral RNA synthesis was lower and the development of the viral nucleocapsid (N) protein was slower than those of MHV-A59. MHV/BHK was 100-fold more resistant to neutralization with the purified soluble recombinant MHV receptor glycoprotein (sMHVR) than was MHV-A59. Pretreatment of 17 Cl 1 cells with anti-MHVR monoclonal antibody CC1 protected the cells from infection with MHV-A59 but only partially protected them from infection with MHV/BHK. Thus, although MHV/BHK could still utilize MHVR as a receptor, its interactions with the receptor were significantly different from those of MHV-A59. To determine whether a hemagglutinin esterase (HE) glycoprotein that could bind the virions to 9-O-acetylated neuraminic acid moieties on the cell surface was expressed by MHV/BHK, an in situ esterase assay was used. No expression of HE activity was detected in 17 Cl 1 cells infected with MHV/BHK, suggesting that this virus, like MHV-A59, bound to cell membranes via its S glycoprotein. MHV/BHK was able to infect cell lines from many mammalian species, including murine (17 Cl 1), hamster (BHK), feline (Fcwf), bovine (MDBK), rat (RIE), monkey (Vero), and human (L132 and HeLa) cell lines. MHV/BHK could not infect dog kidney (MDCK I) or swine testis (ST) cell lines. Thus, in persistently infected murine cell lines that express very low levels of virus receptor MHVR and which also have and may express alternative virus receptors of lesser efficiency, there is a strong selective advantage for virus with altered interactions with receptor (D. S. Chen, M. Asanaka, F. S. Chen, J. E. Shively, and M. M. C. Lai, J. Virol. 71:1688-1691, 1997; D. S. Chen, M. Asanaka, K. Yokomori, F.-I. Wang, S. B. Hwang, H.-P. Li, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 92:12095-12099, 1995; P. Nedellec, G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile, K. V. Holmes, and N. Beauchemin, J. Virol. 68:4525-4537, 1994). Possibly, in coronavirus-infected animals, replication of the virus in tissues that express low levels of receptor might also select viruses with altered receptor recognition and extended host range.
Vaccination against persistent viral infection exacerbates CD4+ T-cell-mediated immunopathological disease
Authors: Hildeman D , Yañez D , Pederson K , Havighurst T , Muller D
Published: January 2020

Lymphocytic choriomeningitis virus (LCMV) infection of normal mice results in a fatal immunopathologic meningitis mediated by CD8+ cytotoxic T lymphocytes (CTL). We have previously shown that female beta2-microglobulin-deficient (beta2m-/-) mice, which are also deficient in CD8+ T cells, are susceptible to LCMV-induced immune-mediated meningitis, characterized by significant weight loss and mortality. This LCMV disease in beta2m-/- mice is mediated by CD4+ T lymphocytes. Our previous studies have also demonstrated that male beta2m-/- mice are less susceptible than female beta2m-/- mice to LCMV-induced, immune-mediated mortality and weight loss. In this report, we show that vaccination of male beta2m-/- mice enhances immunopathology following intracranial infection with LCMV. We observed increased production of gamma interferon (IFN-gamma), an increase in CD4+ CTL precursor frequency, and an increased frequency of IFN-gamma-producing cells from spleen cells of vaccinated male beta2m-/- mice. Vaccinated male beta2m-/- mice also had significantly increased inflammation in the cerebrospinal fluid (CSF), characterized by a large CD4+ T-cell infiltrate. CSF cells from vaccinated mice showed increased production of IFN-gamma on day 7 postchallenge. Neither vaccinated nor control beta2m-/- mice were able to clear virus, and the two groups had similarly high levels of virus early after infection. These results suggest that the magnitude of the early immune response is more important than the level of virus in the brain in determining the outcome of immunopathology in beta2m-/- mice. We show here that vaccination can increase CD4+ T-cell-dependent immunopathology to a persistent viral infection.
Association of herpes simplex virus regulatory protein ICP22 with transcriptional complexes containing EAP, ICP4, RNA polymerase II, and viral DNA requires posttranslational modification by the U(L)13 proteinkinase
Authors: Leopardi R , Ward P L , Ogle W O , Roizman B
Published: January 2020

The expression of herpes simplex virus 1 gamma (late) genes requires functional alpha proteins (gamma1 genes) and the onset of viral DNA synthesis (gamma2 genes). We report that late in infection after the onset of viral DNA synthesis, cell nuclei exhibit defined structures which contain two viral regulatory proteins (infected cell proteins 4 and 22) required for gamma gene expression, RNA polymerase II, a host nucleolar protein (EAP or L22) known to be associated with ribosomes and to bind small RNAs, including the Epstein-Barr virus small nuclear RNAs, and newly synthesized progeny DNA. The formation of these complexes required the onset of viral DNA synthesis. The association of infected cell protein 22, a highly posttranslationally processed protein, with these structures did not occur in cells infected with a viral mutant deleted in the genes U(L)13 and U(S)3, each of which specifies a protein kinase known to phosphorylate the protein.
Human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation mediated by vascular cell adhesion molecule-1: evidence for involvement of cell adhesion molecules in HTLV-1 biology
Authors: Hildreth J E , Subramanium A , Hampton R A
Published: January 2020

While studying the potential role of vascular cell adhesion molecule-1 (VCAM-1) in infection of endothelial cells by human immunodeficiency virus (HIV), we found that VCAM-1 can mediate human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation. Both expression-vector-encoded and endogenously expressed VCAM-1 supported fusion of uninfected cells with HTLV-1-infected cells. Fusion was obtained with cell lines carrying the HTLV-1 genome and expressing viral proteins but not with an HTLV-1-transformed cell line that does not express viral proteins. In clones of VCAM-1-transfected cells, the degree of syncytium formation observed directly reflected the level of VCAM-1 expression. Syncytium formation between HTLV-1-expressing cells and VCAM-1+ cells could be blocked with antiserum against HTLV-1 gp46 and with a monoclonal antibody (MAb) against VCAM-1. Fusion was not blocked by antiserum against HIV or a MAb against VLA-4, the physiological counter-receptor for VCAM-1. The results indicate that VCAM-1 can serve as an accessory molecule or potential coreceptor for HTLV-1-induced cell fusion and provide direct evidence of a role for cell adhesion molecules in the biology of HTLV-1.
Importance of a c-Myb binding site for lymphomagenesis by the retrovirus SL3-3
Authors: Nieves A , Levy L S , Lenz J
Published: January 2020

All murine leukemia viruses (MuLVs) and related type C retroviruses contain a highly conserved binding site for the Ets family of transcription factors within the enhancer sequences in the viral long terminal repeats (LTRs). The T-cell lymphomagenic MuLV SL3-3 (SL3-3) also contains a c-Myb binding site adjacent to the Ets site. The presence of this Myb site distinguishes SL3 from most other MuLVs. We tested the importance of these two sites for the lymphomagenicity of SL3-3. Mutation of the Ets site had little effect on viral pathogenicity, as it only slightly extended the latency period to disease onset. In contrast, mutation of the Myb site strongly inhibited pathogenicity, as only a minority of the inoculated mice developed tumors in the two mouse strains that were tested. All tumors that were induced by either mutant appeared to be lymphomas, and no evidence for reversion of either mutation was detected. The effects of the Ets and Myb site mutations on transcriptional activity of the SL3 LTR were tested by inserting the viral enhancer sequences into a plasmid containing the promoter region of the c-myc gene linked to a reporter gene. Mutation the Myb site almost eliminated enhancer activity in T lymphocytes, while mutation of the Ets site had smaller effects. Thus, the effects of the enhancer mutations on transcriptional activity in T cells paralleled their effects on viral lymphomagenicity. The absence of the c-Myb site in the LTR enhancer of the weakly lymphomagenic MuLV, Akv, likely contributes to the low pathogenicity of this virus relative to SL3-3. However, Moloney MuLV also lacks the Myb site in its LTR, although it induces T-cell lymphomas with a potency similar to that of SL3-3. Thus, it appears that SL3-3 and Moloney MuLV evolved genetic determinants of T-cell lymphomagenicity that are, at least in part, distinct.
Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor
Authors: Matsumoto M , Hsieh T Y , Zhu N , VanArsdale T , Hwang S B , Jeng K S , Gorbalenya A E , Lo S Y , Ou J H , Ware C F , Lai M M
Published: January 2020

Hepatitis C virus (HCV) core protein is a multifunctional protein. We examined whether it can interact with cellular proteins, thus contributing to viral pathogenesis. Using the HCV core protein as a bait to screen a human liver cDNA library in a yeast two-hybrid screening system, we have isolated several positive clones encoding cellular proteins that interact with the HCV core protein. Interestingly, more than half of these clones encode the cytoplasmic domain of lymphotoxin-beta receptor (LT betaR), which is a member of the tumor necrosis factor receptor family. Their binding was confirmed by in vitro glutathione S-transferase fusion protein binding assay and protein-protein blotting assay to be direct and specific. The binding sites were mapped within a 58-amino-acid region of the cytoplasmic tail of LT betaR. The binding site in the HCV core protein was localized within amino acid residues 36 to 91 from the N terminus, corresponding to the hydrophilic region of the protein. In mammalian cells, the core protein was found to be associated with the membrane-bound LT betaR. Since the LT betaR is involved in germinal center formation and developmental regulation of peripheral lymphoid organs, lymph node development, and apoptotic signaling, the binding of HCV core protein to LT betaR suggests the possibility that this viral protein has an immunomodulating function and may explain the mechanism of viral persistence and pathogenesis of HCV.
In vitro model for the nuclear transport of the hepadnavirus genome
Authors: Kann M , Bischof A , Gerlich W H
Published: January 2020

Hepadnaviruses contain a DNA genome, but they replicate via an RNA intermediate, synthesized by the cellular RNA polymerase II in the nucleus of the infected cell. Thus, nuclear transport of the viral DNA is required in the viral life cycle. Protein-free DNA is only poorly imported into the nucleus, so one or more of the viral proteins must be involved in the transport of the viral genome. In order to identify these viral proteins, we purified woodchuck hepadnavirus (WHV) core particles from infected woodchuck liver, isolated WHV DNA, and extracted the covalent complex of viral polymerase from the particles using urea. Intact core particles, the polymerase-DNA complex, or protein-free WHV DNA from core particles was added to digitonin-permeabilized HuH-7 cells, in which the cytosol was substituted by rabbit reticulocyte lysate (RRL) and an ATP-generating system. The distribution of the viral genome was analyzed by semiquantitative PCR or by hybridization in total nuclei, RRL, nuclear membranes, and nucleoplasm. The polymerase-DNA complex was efficiently transported into the nucleus, as indicated by the resistance of the nucleus-associated DNA to a short-term treatment with DNase I of the intact nuclei. The DNA within core particles stayed mainly in the cytosol and remained protected against DNase I. A minor part of the encapsidated DNA was bound to nuclei. It was protected against DNase I but became accessible after disruption of the nuclei. Deproteinized viral DNA completely remained in the cytosol. These data show that the viral polymerase is probably sufficient for mediating the transport of a hepadnavirus genome into the nucleus and that the viral core particles may release the genome at the nuclear membrane.
High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells
Authors: An D S , Koyanagi Y , Zhao J Q , Akkina R , Bristol G , Yamamoto N , Zack J A , Chen I S
Published: January 2020

Gene therapy strategies for humans have been limited by low transduction efficiencies and poor expression of retroviral vectors in differentiated progeny cells carrying the transduced vector. Here we describe a strategy utilizing a cell surface reporter gene, murine thy-1.2, selectable by fluorescence-activated cell sorting (FACS), to achieve higher gene marking efficiencies. Human CD34-positive cells were transduced by a murine retroviral vector bearing the thy-1.2 marker and pseudotyped with vesicular stomatitis virus G protein, followed by FACS to enrich for CD34-positive cells that express Thy-1.2 on the cell surface. Gene marking and expression after differentiation into thymocytes were assessed in a SCID-hu Thy/Liv mouse model for human lymphoid progenitor cell gene therapy. We found that virtually all of the differentiated T-cell progeny were marked with vector sequences. It is of particular importance that reconstitution with the selected cells resulted in expression of Thy-1.2 in up to 71% of donor-derived thymocytes. It is of note that the donor-derived thymocytes that did not express Thy-1.2 still harbored vector thy-1.2 sequences, suggesting repression of transgene expression in some cells during progenitor cell differentiation into thymocytes. These studies provide a proof of concept for efficient expression of transgenes through T-lymphoid differentiation and a potential basis for utilizing similar strategies in human gene therapy clinical trials.
Mutations in the matrix protein of human immunodeficiency virus type 1 inhibit surface expression and virion incorporation of viral envelope glycoproteins in CD4+ T lymphocytes
Authors: Lee Y M , Tang X B , Cimakasky L M , Hildreth J E , Yu X F
Published: January 2020

Highly conserved amino acids in the second helix structure of the human immunodeficiency virus type 1 (HIV-1) MA protein were identified to be critical for the incorporation of viral Env proteins into HIV-1 virions from transfected COS-7 cells. The effects of these MA mutations on viral replication in the HIV-1 natural target cells, CD4+ T lymphocytes, were evaluated by using a newly developed system. In CD4+ T lymphocytes, mutations in the MA domain of HIV-1 Gag also inhibited the incorporation of viral Env proteins into mature HIV-1 virions. Furthermore, mutations in the MA domain of HIV-1 Gag reduced surface expression of viral Env proteins in CD4+ T lymphocytes. The synthesis of gp160 and cleavage of gp160 to gp120 were not significantly affected by MA mutations. On the other hand, the stability of gp120 in MA mutant-infected cells was significantly reduced compared to that in the parental wild-type virus-infected cells. These results suggest that functional interaction between HIV-1 Gag and Env proteins is not only critical for efficient incorporation of Env proteins into mature virions but also important for proper intracellular transport and stable surface expression of viral Env proteins in infected CD4+ T lymphocytes. A single amino acid substitution in MA abolished virus infectivity in dividing CD4+ T lymphocytes without significantly affecting virus assembly, virus release, or incorporation of Gag-Pol and Env proteins, suggesting that in addition to its functional role in virus assembly, the MA protein of HIV-1 also plays an important role in other steps of virus replication.
The CD4-independent tropism of human immunodeficiency virus type 2 involves several regions of the envelope protein and correlates with a reduced activation threshold for envelope-mediated fusion
Authors: Reeves J D , Schulz T F
Published: January 2020

Several human immunodeficiency virus type 2 (HIV-2) strains have been shown to infect some CD4-negative cell lines (P. R. Clapham, A. McKnight, and R. A. Weiss, J. Virol. 66:3531-3537, 1992). Using molecular clones of HIV-2 with a CD4-independent tropism, we have identified critical amino acid residues in the envelope protein which are required for CD4-independent infection. Mutations located immediately upstream of a proposed coiled coil domain in the transmembrane protein (A526T or I528M) and flanking the base of the V4 loop (L378F and K403R) are crucial for the CD4-independent phenotype. Of several mutations conferring a positive charge in V1, V2, and V3, only the change in V3 (Q310K) helped to enhance the CD4-independent phenotype but could not mediate it on its own. These mutations reduce the amount of soluble CD4 required to trigger CD4-independent cell-cell fusion, suggesting that they lower the activation threshold for the fusion process. After binding to cell surface-anchored CD4, a CD4-independent recombinant envelope protein showed an increased binding of anti-envelope protein antibodies, suggesting either an enhanced binding to cell surfaces or more extensive conformational changes in CD4-independent compared to CD4-dependent envelope proteins. The reduced activation threshold of CD4-independent envelope proteins may thus enable them to utilize a membrane molecule for entry which is not as efficient as CD4 in triggering the conformational changes required for the membrane fusion process. CD4-independent HIV-2 variants may be conceptually similar to influenza virus variants capable of fusing at a higher than normal pH (R. S. Daniels, J. C. Downie, J. A. Hay, M. Knossow, J. J. Skehel, M. L. Wang, and D. C. Wiley, Cell 40:431-439, 1985).
Physical and functional interactions between herpes simplex virus immediate-early proteins ICP4 and ICP27
Authors: Panagiotidis C A , Lium E K , Silverstein S J
Published: January 2020

The ordered expression of herpes simplex virus type 1 (HSV-1) genes, during the course of a productive infection, requires the action of the virus immediate-early regulatory proteins. Using a protein interaction assay, we demonstrate specific in vitro protein-protein interactions between ICP4 and ICP27, two immediate-early proteins of HSV-1 that are essential for virus replication. We map multiple points of contact between these proteins. Furthermore, by coimmunoprecipitation experiments, we demonstrate the following. (i) ICP4-ICP27 complexes are present in extracts from HSV-1 infected cells. (ii) ICP27 binds preferentially to less modified forms of ICP4, a protein that is extensively modified posttranslationally. We also demonstrate, by performing electrophoretic mobility shift assays and supershifts with monoclonal antibodies to ICP4 or ICP27, that both proteins are present in a DNA-protein complex with a noncanonical ICP4 binding site present in the HSV thymidine kinase (TK) gene. ICP4, in extracts from cells infected with ICP27-deficient viruses, is impaired in its ability to form complexes with the TK site but not with the canonical site from the alpha4 gene. However, ICP4 is able to form complexes with the TK probe, in the absence of ICP27, when overproduced in mammalian cells or expressed in bacteria. These data suggest that the inability of ICP4 from infected cell extracts to bind the TK probe in the absence of ICP27 does not reflect a requirement for the physical presence of ICP27 in the complex. Rather, they imply that ICP27 is likely to modulate the DNA binding activity of ICP4 by affecting its posttranslational modification status. Therefore, we propose that ICP27, in addition to its established role as a posttranscriptional regulator of virus gene expression, may also modulate transcription either through direct or indirect interactions with HSV regulatory regions, or through its ability to modulate the DNA binding activity of ICP4.
Characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain A59
Authors: Bonilla P J , Hughes S A , Weiss S R
Published: January 2020

The 21.7-kb replicase locus of mouse hepatitis virus strain A59 (MHV-A59) encodes several putative functional domains, including three proteinase domains. Encoded closest to the 5' terminus of this locus is the first papain-like proteinase (PLP-1) (S. C. Baker et al., J. Virol. 67:6056-6063, 1993; H.-J. Lee et al., Virology 180:567-582, 1991). This cysteine proteinase is responsible for the in vitro cleavage of p28, a polypeptide that is also present in MHV-A59-infected cells. Cleavage at a second site was recently reported for this proteinase (P. J. Bonilla et al., Virology 209:489-497, 1995). This new cleavage site maps to the same region as the predicted site of the C terminus of p65, a viral polypeptide detected in infected cells. In this study, microsequencing analysis of the radiolabeled downstream cleavage product and deletion mutagenesis analysis were used to identify the scissile bond of the second cleavage site to between Ala832 and Gly833. The effects of mutations between the P5 and P2' positions on the processing at the second cleavage site were analyzed. Most substitutions at the P4, P3, P2, and P2' positions were permissive for cleavage. With the exceptions of a conservative P1 mutation, Ala832Gly, and a conservative P5 mutation, Arg828Lys, substitutions at the P5, P1, and P1' positions severely diminished second-site proteolysis. Mutants in which the p28 cleavage site (Gly247 / Val248) was replaced by the Ala832 / Gly833 cleavage site and vice versa were found to retain processing activity. Contrary to previous reports, we determined that the PLP-1 has the ability to process in trans at either the p28 site or both cleavage sites, depending on the choice of substrate. The results from this study suggest a greater role by the PLP-1 in the processing of the replicase locus in vivo.
A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle
Authors: Xiao X , Xiao W , Li J , Samulski R J
Published: January 2020

Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (pDD-2) containing only 165 bp of AAV sequence: two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. pDD-2 replication intermediates were characteristic of the AAV replication scheme in which linear monomer, dimer, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV DNA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both rAAV vector production and synthetic plasmid vector delivery.