bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence and drug susceptibility of subtype C reverse transcriptase from human immunodeficiency virus type 1 seroconverters in Zimbabwe
Authors: Shafer R W , Eisen J A , Merigan T C , Katzenstein D A
Published: January 2020

Naturally occurring human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) variability has implications for the success of antiretroviral therapy. We determined the sequence of the polymerase-coding region of RT from virus isolates from 12 Zimbabwean individuals recently infected with HIV-1. The 12 RT sequences differed from the consensus B RT sequence at 10.5% of nucleotides and 5.8% of amino acids. Susceptibility testing of five isolates to zidovudine, didanosine, lamivudine, and nevirapine demonstrated susceptibilities similar to those of wild-type subtype B isolates. Phylogenetic analysis of 40 HIV-1 RT sequences, including the 12 Zimbabwean subtype C sequences, 11 subtype B sequences, and the 17 remaining published non-subtype B sequences showed sufficient intrasubtype RT sequence variation to differentiate subtype A, B, C, and D isolates. Five recently reported subtype C RT sequences from India grouped with the Zimbabwean subtype C sequences but had significantly less intraisolate sequence variation. Both intra- and intersubtype RT comparisons were notable for extraordinarily high ratios of synonymous to nonsynonymous differences. Although substitutions in the HIV-1 RT gene are limited by functional constraints, variation between RT sequences demonstrates phylogenetic relationships that parallel env and gag gene variation.
Inhibition of human immunodeficiency virus replication by the herpes simplex virus virion host shutoff protein
Authors: Hamouda T , McPhee R , Hsia S C , Read G S , Holland T C , King S R
Published: January 2020

The herpes simplex virus (HSV) virion host shutoff gene (vhs) encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis. This ability to inhibit a critical cellular function suggested that vhs could be used as a suicide gene in certain gene therapy applications. To investigate whether vhs might be useful for treatment of AIDS, we tested the ability of both HSV type 1 (HSV-1) and HSV-2 vhs to inhibit replication of human immunodeficiency virus (HIV). Replication of HIV was substantially inhibited when an infectious HIV proviral clone was cotransfected into HeLa cells together with vhs under the control of the cytomegalovirus (CMV) immediate-early promoter. HSV-2 vhs was more active than HSV-1 vhs in these experiments, consistent with previously published studies on these genes. Since expression of vhs from the CMV promoter is essentially unregulated, we also tested the ability of vhs expressed from the HIV long terminal repeat (LTR) promoter to inhibit HIV replication. Wild-type HSV-1 vhs inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene encoding a nonfunctional form of the Vhs protein. Production of Vhs in transfected cells was verified by Western blot assays. A larger amount of Vhs was observed in cells transfected with plasmids expressing vhs from the HIV LTR than from the CMV promoter, consistent with the greater inhibition of HIV replication observed with these constructs. Mutant forms of Vhs were expressed at higher levels than wild-type Vhs, most likely due to the ability of wild-type Vhs to degrade its own mRNA. The strong inhibitory activity of the vhs gene and its unique biological properties make vhs an interesting candidate for use as a suicide gene for HIV gene therapy.
Mutagenesis analysis of the murine leukemia virus matrix protein: identification of regions important for membrane localization and intracellular transport
Authors: Soneoka Y , Kingsman S M , Kingsman A J
Published: January 2020

We have created two sets of substitution mutations in the Moloney murine leukemia virus (Mo-MuLV) matrix protein in order to identify domains involved in association with the plasma membrane and in incorporation of the viral envelope glycoproteins into virus particles. The first set of mutations was targeted at putative membrane-associating regions similar to those of the human immunodeficiency virus type 1 matrix protein, which include a polybasic region at the N terminus of the Mo-MuLV matrix protein and two regions predicted to form beta strands. The second set of mutations was created within hydrophobic residues to test for the production of virus particles lacking envelope proteins, with the speculation of an involvement of the membrane-spanning region of the envelope protein in incorporation into virus particles. We have found that mutation of the N-terminal polybasic region redirected virus assembly to the cytoplasm, and we show that tryptophan residues may also play a significant role in the intracellular transport of the matrix protein. In total, 21 mutants of the Mo-MuLV matrix protein were produced, but we did not observe any mutant virus particles lacking the envelope glycoproteins, suggesting that a direct interaction between the Mo-MuLV matrix protein and envelope proteins either may not exist or may occur through multiple redundant interactions.
The range and distribution of murine central nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus 1
Authors: Markovitz N S , Baunoch D , Roizman B
Published: January 2020

Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the gamma(1)34.5- gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). To investigate the host range and distribution of infected cells in the CNS of mice, 4- to 5-week-old mice were inoculated stereotaxically into the caudate/putamen with 3 x 10(5) PFU of the gamma(1)34.5- virus R3616. Four-micrometer-thick sections of mouse brains removed on day 3, 5, or 7 after infection were reacted with a polyclonal antibody directed primarily to structural proteins of the virus and with antibodies specific for neurons, astrocytes, or oligodendrocytes. This report shows the following: (i) most of the tissue damage caused by R3616 was at the site of injection, (ii) the virus spread by retrograde transport from the site of infection to neuronal cell nuclei at distant sites and to ependymal cells by cerebrospinal fluid, (iii) the virus infected neurons, astrocytes, oligodendrocytes, and ependymal cells and hence did not discriminate among CNS cells, (iv) viral replication in some neurons could be deduced from the observation of infected astrocytes and oligodendrocytes at distant sites, and (v) infected cells were being efficiently cleared from the nervous system by day 7 after infection. We conclude that the gamma(1)34.5- attenuation phenotype is reflected in a gross reduction in the ability of the virus to replicate and spread from cell to cell and is not due to a restricted host range. The block in viral replication appears to be a late event in viral replication.
Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells
Authors: Qing K , Bachelot T , Mukherjee P , Wang X S , Peng L , Yoder M C , Leboulch P , Srivastava A
Published: January 2020

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.
Determinants of the human immunodeficiency virus type 1 p15NC-RNA interaction that affect enhanced cleavage by the viral protease
Authors: Sheng N , Pettit S C , Tritch R J , Ozturk D H , Rayner M M , Swanstrom R , Erickson-Viitanen S
Published: January 2020

During human immunodeficiency virus type 1 (HIV-1) virion assembly, cleavage of the Gag precursor by the viral protease results in the transient appearance of a nucleocapsid-p1-p6 intermediate product designated p15NC. Utilizing the p15NC precursor protein produced with an in vitro transcription-translation system or purified after expression in Escherichia coli, we have demonstrated that RNA is required for efficient cleavage of HIV p15NC. Gel mobility shift and nitrocellulose filter binding experiments indicate that purified p15NC protein specifically binds its corresponding mRNA with an estimated Kd of 1.5 nM. Binding was not affected by the presence or absence of zinc or EDTA. Moreover, mutagenesis of the cysteine residues within either of the two Cys-His arrays had no effect on RNA binding or on RNA-dependent cleavage by the viral protease. In contrast, decreased binding of RNA and diminished susceptibility to cleavage in vitro were observed with p15NC-containing mutations in one or more residues within the triplet of basic amino acids present in the region between the two zinc fingers. In addition, we found that 21- to 24-base DNA and RNA oligonucleotides of a particular sequence and secondary structure could substitute for p15 RNA in the enhancement of p15NC cleavage. Virus particles carrying a mutation in the triplet of NC basic residues (P3BE) show delayed cleavage of p15NC and a defect in core formation despite the eventual appearance of fully processed virion protein. These results define determinants of the p15NC-RNA interaction that lead to enhanced protease-mediated cleavage and demonstrate the importance of the triplet of basic residues in formation of the virus core.
Sequence analysis of the hepatitis C virus genome recovered from serum, liver, and peripheral blood mononuclear cells of infected chimpanzees
Authors: Shimizu Y K , Igarashi H , Kanematu T , Fujiwara K , Wong D C , Purcell R H , Yoshikura H
Published: January 2020

Of 13 different strains of hepatitis C virus (HCV) in the inoculum used, only 1 persisted in human lymphocyte cell lines infected in vitro (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). To determine whether that particular strain (designated H1-2) has a tropism for lymphocytes in vivo, we sequenced hypervariable region 1 (HVR1) of the genome of HCV recovered from the sera, livers, and peripheral blood mononuclear cells (PBMC) of chimpanzees infected with plasma H77, the same inoculum used for the in vitro studies. In the PBMC collected from two chimpanzees during the early phase of infection, H1-2 was detected as the only or predominant HVR1 sequence. H1-2 was also detected in PBMC obtained during persistent infection from a chimpanzee that had been treated with immunosuppressants. From the livers of these chimpanzees, two to six different strains were recovered but H1-2 was not detected. Thus, H1-2 appeared to have an affinity for lymphocytes not only in vitro but also in vivo. In samples collected from a chimpanzee after 6 years of infection, however, such tissue compartmentalization of the HCV genome was not observed; a single strain became predominant in the serum, liver, and PBMC. An HCV strain capable of replicating in both the liver and PBMC probably emerged during in vivo replication and persisted.
Enhanced mucosal and systemic immune responses to intestinal reovirus infection in beta2-microglobulin-deficient mice
Authors: Major A S , Cuff C F
Published: January 2020

Enteric infection of mice with respiratory enteric orphan virus (reovirus) type 1, strain Lang elicits both humoral and cellular immune responses. To investigate the role of CD8+, alpha/beta T-cell receptor (TCR)+ T cells in mucosal immunity to an enteric pathogen, we examined immune responses and viral clearance following enteric reovirus infection in C57BL/6, B6129F2, and beta2-microglobulin-deficient (beta2m-/-) mice. Analysis of Peyer's patch and lamina propria culture supernatants revealed a two- to threefold increase in levels of reovirus-specific immunoglobulin A in beta2m-/- mice compared to normal controls. These data corresponded to a similar increase in the frequency of virus-specific immunoglobulin A-producing cells in Peyer's patches and lamina propria and an increase in immunoglobulin G-producing cells in spleens from beta2m-/- mice compared to controls. These increased humoral immune responses were not due to a difference in B-cell populations because cell counts and flow cytometric analyses showed that beta2m-/- and control mice had similar numbers and percentages of B cells in mucosal and systemic tissues. Analysis of cytokine message by reverse transcriptase-PCR 5 and 10 days after infection revealed no difference in message level for transforming growth factor beta, gamma interferon, interleukin-4, interleukin-5, or interleukin-6 for all mouse strains. Virus tissue titers determined by plaque assay at 5 and 10 days after infection demonstrated that beta2m-/- mice cleared reovirus from the small intestines with the same efficiency as control mice. Collectively, these data suggest that CD8+, alpha/beta TCR+ T cells may regulate mucosal and systemic humoral immune responses to oral infection with reovirus.
The VP16 paradox: herpes simplex virus VP16 contains a long-range activation domain but within the natural multiprotein complex activates only from promoter-proximal positions
Authors: Hagmann M , Georgiev O , Schaffner W
Published: January 2020

Removal of core promoter elements like the TATA box converts several regulatory upstream regions of viral and cellular genes into classical enhancers, i.e., cis-regulatory elements capable of activating transcription over long distances in an orientation-independent manner. This is not the case with herpes simplex virus (HSV) immediate-early gene promoters, which are strongly induced by the viral transactivator VP16 (Vmw65, alphaTIF, ICP25) complexed with the cellular factors Oct-1 and HCF. Here we report that the VP16 complex can readily bring about strong activation from a promoter-proximal position but fails to induce transcription from a distal downstream enhancer position. This is in striking contrast to results obtained with GAL fusion proteins: in this context, the C-terminal "general" activation domain of VP16 activates transcription to high levels over long distances. Thus, this paradoxical behavior suggests that the VP16 activation domain is not accessible to the transcription machinery when the VP16-Oct-1-HCF complex is bound in a remote position. Only upon specific interactions in a promoter-proximal position, perhaps with the basal transcription factors, can transcription be strongly induced. In agreement with such a proposed mechanism, VP16 proteins to which a heterologous general activation domain has been added strongly activate transcription from a downstream position. The biological role of this unexpected and sophisticated mechanism is most probably a limitation of the VP16 activity to the associated immediate-early genes, without undesired long-range effects on other viral promoters within the tightly packed HSV genome.
High-efficiency incorporation of functional influenza virus glycoproteins into recombinant vesicular stomatitis viruses
Authors: Kretzschmar E , Buonocore L , Schnell M J , Rose J K
Published: January 2020

We derived recombinant vesicular stomatitis virus (VSV) expressing either influenza virus hemagglutinin (HA) or neuraminidase (NA) glycoproteins from extra genes inserted in the viral genome. The HA protein was expressed from a site downstream of the VSV glycoprotein (G) gene, while NA protein was expressed from a site upstream of the VSV G gene. The HA protein was expressed at lower levels than the VSV G protein, while the NA protein was expressed at higher levels, as expected from the gradient of VSV transcription that follows the gene order. The HA and NA proteins were transported to the cell surface and were functional as demonstrated by hemadsorption, hemolysis, and NA assays. Biochemical analysis showed that both HA and NA proteins were incorporated into VSV particles at high levels, although there was a preference for incorporation of the VSV G protein over either of the influenza virus proteins. Immunoelectron microscopy of the recombinants showed that the particles derived from the recombinants were mosaics carrying both the VSV G protein and the influenza virus membrane glycoproteins. These results extend earlier studies showing incorporation of the cellular glycoprotein CD4 and two other viral glycoproteins into VSV particles. Our results indicate that there is significant space in the VSV membrane that can accommodate foreign membrane proteins and that the foreign protein can represent as much as 35% of the total protein in the viral envelope. Incorporation of foreign proteins into VSV virions can, in many cases, occur passively in the absence of specific incorporation signals.
Identification of sequences downstream of the primer binding site that are important for efficient replication of human immunodeficiency virus type 1
Authors: Li X , Liang C , Quan Y , Chandok R , Laughrea M , Parniak M A , Kleiman L , Wainberg M A
Published: January 2020

Reverse transcription of retroviruses is initiated from an 18-nucleotide (nt) primer binding site (PBS), located within the 5' region of viral genomic RNA, to which the host cell-derived tRNA primer is annealed and also involves viral genomic sequences outside the PBS. We constructed proviral DNA clones of human immunodeficiency virus (HIV) that had selective deletions of either a 7-nt segment found immediately downstream of the PBS or an extended nontranslated 54-nt stretch located immediately downstream of the PBS and containing the aforementioned 7-nt segment. Synthesis of minus-strand strong-stop DNA was assessed with MT-4 cells infected with viruses derived from COS-7 cells that had been transfected with these various constructs. We found that similar levels of minus-strand strong-stop DNA as well as DNA produced after template switching were expressed in MT-4 cells infected with COS-7-derived wild-type viruses or with viruses that had the 7-nt segment deleted. In contrast, significantly lower levels of viral DNA were detected in MT-4 cells after infection with viruses that had deletions of the 54-nt stretch. Furthermore, the molecular clone containing the 7-nt deletion was able to replicate with wild-type kinetics, while that containing the 54-nt deletion displayed a significantly diminished capacity in this regard. Further deletion analysis showed that a 16-nt segment at the 3' end of this 54-nt segment was largely responsible for these effects. We also conducted studies to determine levels of viral mRNA in COS-7 cells that had been transfected with equivalent amounts of DNA derived from either a wild-type HIV construct or our various deletion mutants. In the case of transfections performed with the 7-nt deletion mutant and wild-type HIV DNA, high levels of viral mRNA transcripts were detected, which was not the case for the 54 nt-deletion mutant. However, these various mRNAs possessed similar stabilities, as shown through studies in which transcript formation was arrested by treatment of cells with actinomycin D. Thus, the 54-nt segment of 5' nontranslated RNA, located downstream of the PBS, is involved in efficient expression of each of viral DNA, mRNA, and infectious virus.
A role for natural simian immunodeficiency virus and human immunodeficiency virus type 1 nef alleles in lymphocyte activation
Authors: Alexander L , Du Z , Rosenzweig M , Jung J U , Desrosiers R C
Published: January 2020

A T-lymphoid cell line termed 221 was derived from a rhesus monkey infected with herpesvirus saimiri. Growth of 221 cells was dependent on the addition of interleukin-2 (IL-2) to the culture medium. In the absence of IL-2, 221 cells arrested in G0-G1 but did not die. Simian immunodeficiency virus (SIV) replicated efficiently in IL-2-stimulated 221 cells whether or not the nef gene was present. In the absence of IL-2, nef-containing SIV replicated 8 to 100 times more efficiently in 221 cells than did the same virus lacking nef. nef-containing virus preferentially stimulated the production of IL-2 from 221 cells. HIV-1 nef and v-ras genes, but not the c-ras gene, were shown to substitute functionally for SIV nef when tested as recombinant viruses in this assay system. These results demonstrate a role for natural nef in causing lymphoid cell activation, and they provide a system for delineating the biochemical mechanisms responsible for this activation.
In vitro analysis of the E1A-homologous sequences of RIZ
Authors: Buyse I M , Huang S
Published: January 2020

The RIZ (G3B/MTB-Zf) gene was first isolated based on its ability to bind to the retinoblastoma protein (Rb). An acidic, approximately 100-amino-acid region around the Rb-binding motif of RIZ has structural and antigenic similarity to the conserved sequences of the E1A viral oncogene. We show here that this region interacts specifically with the E1A-binding domain of Rb. This interaction could be disrupted by E1A or by a peptide of RIZ homologous to the CR2 motif of E1A which is involved in binding to Rb family proteins. Also like E1A, RIZ can form a ternary complex with Rb and E2F1. Despite this similarity to E1A, however, RIZ could not bind to the Rb family proteins p107 and p130 in vitro. The data show that the RIZ CR2 motif can mediate differential binding to Rb family proteins. We also mapped the shared antigenic determinant between RIZ and E1A to a conserved sequence, designated CE1, which is located in the C terminus of E1A. Unlike that of ETA, the CE1 motif of RIZ is located next to the CR2 motif. Despite this proximity, CE1 and CR2 appear to act independently. The data show similarities as well as differences between the homologous sequences of RIZ and E1A and contribute to an understanding of the biochemistry of these proteins.
Poliovirus protein 2BC increases cytosolic free calcium concentrations
Authors: Aldabe R , Irurzun A , Carrasco L
Published: January 2020

Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis.
Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor
Authors: Hill C M , Deng H , Unutmaz D , Kewalramani V N , Bastiani L , Gorny M K , Zolla-Pazner S , Littman D R
Published: January 2020

Several members of the chemokine receptor family have recently been identified as coreceptors, with CD4, for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. In this report, we show that the envelope glycoproteins of several strains of HIV-2 and simian immunodeficiency virus (SIV) employ the same chemokine receptors for infection. Envelope glycoproteins from HIV-2 use CCR5 or CXCR4, while those from several strains of SIV use CCR5. Our data indicate also that some viral envelopes can use more than one coreceptor for entry and suggest that some of these coreceptors remain to be identified. To further understand how different envelope molecules use CCR5 as an entry cofactor, we show that soluble purified envelope glycoproteins (SU component) from CCR5-tropic HIV-1, HIV-2, and SIV can compete for binding of iodinated chemokine to CCR5. The competition is dependent on binding of the SU glycoprotein to cell surface CD4 and implies a direct interaction between envelope glycoproteins and CCR5. This interaction is specific since it is not observed with SU glycoprotein from a CXCR4-tropic virus or with a chemokine receptor that is not competent for viral entry (CCR1). For HIV-1, the interaction can be inhibited by antibodies specific for the V3 loop of SU. Soluble CD4 was found to potentiate binding of the HIV-2 ST and SIVmac239 envelope glycoproteins to CCR5, suggesting that a CD4-induced conformational change in SU is required for subsequent binding to CCR5. These data suggest a common fundamental mechanism by which structurally diverse HIV-1, HIV-2, and SIV envelope glycoproteins interact with CD4 and CCR5 to mediate viral entry.
The potent enhancer activity of the polycythemic strain of spleen focus-forming virus in hematopoietic cells is governed by a binding site for Sp1 in the upstream control region and by a unique enhancer core motif, creating an exclusive target for PEBP/CBF
Authors: Baum C , Itoh K , Meyer J , Laker C , Ito Y , Ostertag W
Published: January 2020

The polycythemic strain of the spleen focus-forming virus (SFFVp) contains the most potent murine retroviral enhancer configuration known so far for gene expression in myeloerythroid hematopoietic cells. In the present study, we mapped two crucial elements responsible for the high activity of the SFFVp enhancer to an altered upstream control region (UCR) containing a GC-rich motif (5'-GGGCGGG-3') and to a unique enhancer core (5'-TGCGGTC-3'). Acquisition of these motifs accounts for half of the activity of the complete retroviral enhancer in hematopoietic cells, irrespective of the developmental stage or lineage. Furthermore, the UCR motif contains the major determinant for the enhancer activity of SFFVp in embryonic stem (ES) cells. Using electrophoretic mobility shift assays, we show that the UCR of SFFVp, but not of Friend murine leukemia virus, is targeted by the ubiquitous transcriptional activator, Sp1. The core motif of SFFVp creates a specific and high-affinity target for polyomavirus enhancer binding protein/core binding factor (PEBP/CBF) and excludes access of CAAT/enhancer binding protein. Cotransfection experiments with ES cells imply that PEBP/CBF cooperates with the neighboring element, LVb (the only conserved Ets consensus in the SFFVp enhancer), and that the Sp1 motif in the UCR stimulates transactivation through the Ets-PEBP interaction. Putative secondary structures of the retroviral enhancers are proposed based on these data.
In vitro study of the NS2-3 protease of hepatitis C virus
Authors: Pieroni L , Santolini E , Fipaldini C , Pacini L , Migliaccio G , La Monica N
Published: January 2020

Processing at the C terminus of the NS2 protein of hepatitis C virus (HCV) is mediated by a virus-encoded protease which spans most of the NS2 protein and part of the NS3 polypeptide. In vitro cotranslational cleavage at the 2-3 junction is stimulated by the presence of microsomal membranes and ultimately results in the membrane insertion of the NS2 polypeptide. To characterize the biochemical properties of this viral protease, we have established an in vitro assay whereby the NS2-3 protease of HCV BK can be activated posttranslationally by the addition of detergents. The cleavage proficiency of several deletion and single point mutants was the same as that observed with microsomal membranes, indicating that the overall sequence requirements for proper cleavage at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to the type of detergent used and its concentration. Also, the incubation temperature affected the cleavage at the 2-3 junction. The autoproteolytic activity of the NS2-3 protease could be inhibited by alkylating agents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA inhibition of NS2-3 cleavage could be reversed, at least in part, by the addition of ZnCl2 and CdCl2. Among the common protease inhibitors tested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhibitor inactivated the NS2-3 protease. By means of gel filtration analysis, it was observed that the redox state of the reaction mixture greatly influenced the processing efficiency at the 2-3 site and that factors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required for efficient processing at this site. Thus, the in vitro assay should allow further characterization of the biochemical properties of the NS2-3 protease of HCV and the identification of host components that contribute to the efficient processing at the 2-3 junction.
Role of preterminal protein processing in adenovirus replication
Authors: Webster A , Leith I R , Nicholson J , Hounsell J , Hay R T
Published: January 2020

Preterminal protein (pTP), the protein primer for adenovirus DNA replication, is processed at two sites by the virus-encoded protease to yield mature terminal protein (TP). Here we demonstrate that processing to TP, via an intermediate (iTP), is conserved in all serotypes sequenced to date; and in determining the sites cleaved in Ad4 pTP, we extend the previously published substrate specificity of human adenovirus proteases to include a glutamine residue at P4. Furthermore, using monoclonal antibodies raised against pTP, we show that processing to iTP and TP are temporally separated in the infectious cycle, with processing to iTP taking place outside the virus particles. In vitro and in vivo studies of viral DNA replication reveal that iTP can act as a template for initiation and elongation and argue against a role for virus-encoded protease in switching off DNA replication. Virus DNA with TP attached to its 5' end (TP-DNA) has been studied extensively in in vitro DNA replication assays. Given that in vivo pTP-DNA, not TP-DNA, is the template for all but the first round of replication, the two templates were compared in vitro and shown to have different properties. Immunofluorescence studies suggest that a region spanning the TP cleavage site is involved in defining the subnuclear localization of pTP. Therefore, a likely role for the processing of pTP-DNA is to create a distinct template for early transcription (TP-DNA), while the terminal protein moiety, be it TP or pTP, serves to guide the template to the appropriate subcellular location through the course of infection.
The hepatitis C virus NS4A protein: interactions with the NS4B and NS5A proteins
Authors: Lin C , Wu J W , Hsiao K , Su M S
Published: January 2020

Hepatitis C virus encodes a large polyprotein precursor that is proteolytically processed into at least 10 distinct products, in the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase encoded in the N-terminal 181 residues of the NS3 nonstructural protein is responsible for cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region. NS4A, a 54-residue nonstructural protein which forms a stable complex with the NS3 proteinase, is required as a cofactor for cleavage at the 3/4A and 4B/5A sites and enhances processing at the 4A/4B and 5A/5B sites. Recently reported crystal structures demonstrated that NS4A forms an integral part of the NS3 serine proteinase. In this report, we present evidence that NS4A forms a nonionic-detergent-stable complex with the NS4B5A polyprotein substrate, which may explain the requirement of NS4A for the 4B/5A cleavage. Isoleucine-29 of NS4A, which has been previously shown to be essential for its proteinase cofactor activity and formation of the NS3 complex, was found to be important for the interaction between NS4A and the NS4B5A substrate. In addition, two more hydrophobic residues in the NS4A central region (valine-23 and isoleucine-25) were also shown to be essential for the cofactor activity and for the interaction with either the NS3 proteinase or the NS4B5A polyprotein substrate. Finally, the possible mechanisms by which these viral proteins interact with each other are discussed.
A 437-base-pair deletion at the beginning of the latency-associated transcript promoter significantly reduced adrenergically induced herpes simplex virus type 1 ocular reactivation in latently infected rabbits
Authors: Hill J M , Garza H H , Su Y H , Meegalla R , Hanna L A , Loutsch J M , Thompson H W , Varnell E D , Bloom D C , Block T M
Published: January 2020

In this study we used a herpes simplex virus type 1 (HSV-1) deletion mutant to identify a segment of the genome necessary for epinephrine-induced reactivation in the rabbit eye model of herpetic recurrent disease. In HSV-1 latently infected neural tissue, the only abundant viral products are the latency-associated transcripts (LATs). At least one promoter of LAT has been identified, and mutations in the LAT domain have been used to investigate HSV-1 reactivation. We used an ocular rabbit model of epinephrine-induced HSV-1 reactivation to study the effects of deleting a 437-bp region beginning 796 bp upstream of the LAT CAP site. Specifically, the 437-bp deletion is located between genomic positions 118006 and 118443 of the parent 17Syn+, and the construct is designated 17 delta S/N. This region also controls a portion of the genome encoding two transcripts (1.1 and 1.8 kb) from the LAT domain. A rescuant, 17 delta S/N-Res, was constructed from 17 delta S/N. Following ocular infection, all three viruses produced similar acute dendritic lesions in rabbits. Five weeks after infection, rabbits received transcorneal iontophoresis of epinephrine. The parent, 17Syn+, and the rescuant, 17 delta S/N-Res, underwent a high frequency of HSV-1 ocular reactivation as determined by recovery of infectious virus in the tear film. Rabbits infected with 17 delta S/N had a significantly lower frequency of ocular reactivation. Analysis of the trigeminal ganglia from all three groups of latently infected rabbits revealed (i) similar amounts of HSV DNA (genomic equivalents), (ii) accumulation of 2.0- and 1.45-kb LATs, and (iii) explant reactivation at the same high frequency. Therefore, these studies indicate that the 437-bp deleted region in 17 delta S/N is essential for epinephrine-induced reactivation and could implicate the 1.1- and 1.8-kb transcripts in the mechanisms controlling HSV-1 reactivation.
The Epstein-Barr virus-induced Ca2+/calmodulin-dependent kinase type IV/Gr promotes a Ca(2+)-dependent switch from latency to viral replication
Authors: Chatila T , Ho N , Liu P , Liu S , Mosialos G , Kieff E , Speck S H
Published: January 2020

The switch from latency to viral replication in Epstein-Barr virus (EBV)-transformed human B cells is mediated by Zta, the protein product of immediate-early EBV gene BZLF1. BZLF1 transcription is normally suppressed in EBV-transformed B cells but can be induced in some cell lines upon ligation of surface immunoglobulin by mechanisms that include the activation of Ca(2+)-dependent signaling pathways. The multifunctional Ca2+/calmodulin-dependent kinase type IV/Gr (CaMKIV/Gr) is normally absent in primary human B cells, but its expression is induced by the EBV oncoprotein LMP1 in the course of B-cell growth transformation by EBV. In this study, we demonstrate that activated CaMKIV/Gr induces transcription from the BZLF1 promoter and upregulates the expression of Zta in permissive cells. Transcriptional activation of the BZLF1 promoter by CaMKIV/Gr is dependent on the CREB/AP1 binding element ZII and is greatly augmented by the Ca2+/calmodulin-dependent phosphatase calcineurin. These results outline a virus-regulated mechanism involving CaMKIV/Gr which promotes transition from latency to productive viral replication in response to Ca(2+)-mobilizing extracellular signals.
Epstein-Barr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation
Authors: Harada S , Kieff E
Published: January 2020

Epstein-Barr virus (EBV) nuclear proteins EBNA-LP and EBNA-2 are the first two proteins expressed in latent infection of primary B lymphocytes. EBNA-2 is essential for lymphocyte transformation, and EBNA-LP is at least critical. While EBNA-2 activates specific viral and cellular promoters, EBNA-LP's role has been obscure. We now show that EBNA-LP stimulates EBNA-2 activation of the LMP1 promoter and of the LMP1/LMP2B bidirectional transcriptional regulatory element. EBNA-LP alone has only a negative effect. EBNA-LP also stimulates EBNA-2 activation of a multimerized regulatory element from the BamC EBNA promoter. Since both viral regulatory elements can bind the EBNA-2-associated cell protein RBPJ kappa, consensus RBPJ kappa binding sites were positioned upstream of the herpes simplex virus type 1 thymidine kinase promoter and were found to be sufficient for EBNA-LP and EBNA-2 coactivation. EBNA-LP strongly stimulated activation of an adenovirus E1b promoter with upstream Gal4 binding sites by a Gal4 DNA binding domain/ EBNA-2 acidic domain fusion protein, indicating that EBNA-LP coactivation requires only the EBNA-2 acidic domain to be localized near a promoter. The EBNA-LP stimulatory activity resides in the amino-terminal 66-amino-acid repeat domain. The carboxyl-terminal unique 45 amino acids appear to regulate EBNA-LP's effects. The first 11 amino acids of the 45 have a strong negative effect, while the last 10 are critical for the ability of the last 34 to relieve the negative effect. These results indicate that EBNA-LP's critical role in EBV-mediated cell growth transformation is in stimulating (and probably regulating) EBNA-2-mediated transcriptional activation.
Direct repeats of the herpes simplex virus a sequence promote nonconservative homologous recombination that is not dependent on XPF/ERCC4
Authors: Yao X D , Matecic M , Elias P
Published: January 2020

We have examined mechanisms of recombination in mammalian cells infected with herpes simplex virus type 1 (HSV-1). Amplification of plasmids containing a viral origin of replication, oriS, in cells superinfected with HSV-1 revealed that linear DNA could be efficiently converted to templates for replication. Two distinct pathways were observed: imprecise end joining and nonconservative homologous recombination. We noted that direct repeats of the viral a sequence promoted efficient nonconservative homologous recombination in BHK cells as well as human repair-proficient 1BR.3N cells and xeroderma pigmentosum group F (XP-F) cells. The reaction gave rise to functional a sequences supporting the formation of defective viruses. It did not seem to proceed by single-strand annealing since it occurred in the absence of XPF/ERCC4, the mammalian homolog of the Rad1 endonuclease from Saccharomyces cerevisiae. In contrast, direct repeats of a 161-bp nonviral sequence did not take part in nonconservative homologous recombination in XP-F cells. Our results suggest that homologous recombination may be involved in the circularization of viral genomes. Furthermore, they demonstrate that amplification of recombination products supported by HSV-1 allows a direct examination of pathways for double-strand-break repair in human cells.
Poliovirus Sabin type 1 neutralization epitopes recognized by immunoglobulin A monoclonal antibodies
Authors: Fiore L , Ridolfi B , Genovese D , Buttinelli G , Lucioli S , Lahm A , Ruggeri F M
Published: January 2020

Immunity to poliomyelitis is largely dependent on humoral neutralizing antibodies, both after natural (wild virus or vaccine) infection and after inactivated poliovirus vaccine inoculation. Although the production of local secretory immunoglobulin A (IgA) antibody in the gut mucosa may play a major role in protection, most of information about the antigenic determinants involved in neutralization of polioviruses derives from studies conducted with humoral monoclonal antibodies (MAbs) generated from parenterally immunized mice. To investigate the specificity of the mucosal immune response to the virus, we have produced a library of IgA MAbs directed at Sabin type 1 poliovirus by oral immunization of mice with live virus in combination with cholera toxin. The epitopes recognized by 13 neutralizing MAbs were characterized by generating neutralization-escape virus mutants. Cross-neutralization analysis of viral mutants with MAbs allowed these epitopes to be divided into four groups of reactivity. To determine the epitope specificity of MAbs, virus variants were sequenced and the mutations responsible for resistance to the antibodies were located. Eight neutralizing MAbs were found to be directed at neutralization site N-AgIII in capsid protein VP3; four more MAbs recognized site N-AgII in VP1 or VP2. One IgA MAb selected a virus variant which presented a unique mutation at amino acid 138 in VP2, not previously described. This site appears to be partially related with site N-AgII and is located in a loop region facing the VP2 N-Ag-II loop around residue 164. Only 2 of 13 MAbs proved able to neutralize the wild-type Mahoney strain of poliovirus. The IgA antibodies studied were found to be produced in the dimeric form needed for recognition by the polyimmunoglobulin receptor mediating secretory antibody transport at the mucosal level.
Differential divalent cation requirements uncouple the assembly and catalytic reactions of human immunodeficiency virus type 1 integrase
Authors: Hazuda D J , Felock P J , Hastings J C , Pramanik B , Wolfe A L
Published: January 2020

Previous in vitro analyses have shown that the human immunodeficiency virus type 1 (HIV-1) integrase uses either manganese or magnesium to assemble as a stable complex on the donor substrate and to catalyze strand transfer. We now demonstrate that subsequent to assembly, catalysis of both 3' end processing and strand transfer requires a divalent cation cofactor and that the divalent cation requirements for assembly and catalysis can be functionally distinguished based on the ability to utilize calcium and cobalt, respectively. The different divalent cation requirements manifest by these processes are exploited to uncouple assembly and catalysis, thus staging the reaction. Staged 3' end processing and strand transfer assays are then used in conjunction with exonuclease III protection analysis to investigate the effects of integrase inhibitors on each step in the reaction. Analysis of a series of related inhibitors demonstrates that these types of compounds affect assembly and not either catalytic process, therefore reconciling the apparent disparate results obtained for such inhibitors in assays using isolated preintegration complexes. These studies provide evidence for a distinct role of the divalent cation cofactor in assembly and catalysis and have implications for both the identification and characterization of integrase inhibitors.
Varicella-zoster virus open reading frame 4 encodes an immediate-early protein with posttranscriptional regulatory properties
Authors: Defechereux P , Debrus S , Baudoux L , Rentier B , Piette J
Published: January 2020

Varicella-zoster virus (VZV) encodes four putative immediate-early proteins based on sequence homology with herpes simplex virus type 1: the products of ORF4, -61, -62, and -63. Until now, only two VZV proteins have been described as being truly expressed with immediate-early kinetics (IE62 and IE63). The ORF4-encoded protein can stimulate gene expression either alone or in synergy with the major regulatory protein IE62. Making use of a sequential combination of transcription and protein synthesis inhibitors (actinomycin D and cycloheximide, respectively), we demonstrated the immediate-early nature of the ORF4 gene product, which can thus be named IE4. The fact that IE4 is expressed with kinetics similar to that of IE62 further underlines the possible cooperation between these two VZV proteins in gene expression. Analysis of the IE4-mediated autologous or heterologous viral gene expression at the mRNA levels clearly indicated that IE4 may have several mechanisms of action. Activation of the two VZV genes tested could occur partly by a posttranscriptional mechanism, since increases in chloramphenicol acetyltransferase (CAT) mRNA levels do not account for the stimulation of CAT activity. On the other hand, stimulation of the human immunodeficiency virus type 1 long terminal repeat- or the cytomegalovirus promoter-associated CAT activity is correlated with an increase in the corresponding CAT mRNA.
Effect of Inactivation by Hydroxylamine on Early Functions of Poliovirus
Authors: Borgert Karin , Koschel Klaus , Täuber Helmut , Wecker Eberhard
Published: January 2020

Evidence is presented that poliovirus particles with a single lethal hit by hydroxylamine do not induce in host cells either inhibition of cellular protein synthesis or viral ribonucleic acid (RNA) replication. The RNA of these viruses is not replicated even if the cells are simultaneously infected with both active and inactivated viruses. The damaged viral RNA seems to have lost both its template function and its function in the translation of normal viral proteins.