bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the Effect of Natural Sequence Variation in Tat and in Cyclin T on the Formation and RNA Binding Properties of Tat-Cyclin T Complexes
Authors: Bieniasz Paul D. , Grdina Therese A. , Bogerd Hal P. , Cullen Bryan R.
Published: December 2019

The biological activity of the human immunodeficiency virus type 1 (HIV-1) Tat (Tat1) transcriptional activator requires the recruitment of a Tat1-CyclinT1 (CycT1) complex to the TAR RNA target encoded within the viral long terminal repeat (LTR). While other primate immunodeficiency viruses, such as HIV-2 and mandrill simian immunodeficiency virus (SIV
mnd
), also encode Tat proteins that activate transcription via RNA targets, these proteins differ significantly, both from each other and from Tat1, in terms of their ability to activate transcription directed by LTR promoter elements found in different HIV and SIV isolates. Here, we show that CycT1 also serves as an essential cofactor for HIV-2 Tat (Tat2) and SIV
mnd
Tat (Tat-M) function. Moreover, the CycT1 complex formed by each Tat protein displays a distinct RNA target specificity that accurately predicts the level of activation observed with a particular LTR. While Tat2 and Tat-M share the ability of Tat1 to bind to CycT1, they differ from Tat1 in that they are also able to bind to the related but distinct CycT2. However, the resultant Tat-CycT2 complexes fail to bind TAR and are therefore abortive. Surprisingly, mutation of a single residue in CycT2 (asparagine 260 to cysteine) rescues the ability of CycT2 to bind Tat1 and also activates not only TAR binding by all three Tat-CycT2 complexes but also Tat function. Therefore, the RNA target specificity of different Tat-CycT1 complexes is modulated by natural sequence variation in both the viral Tat transcriptional activator and in the host cell CycT molecule recruited by Tat. Further, the RNA target specificity of the resultant Tat-CycT1 complex accurately predicts the ability of that complex to activate transcription from a given LTR promoter element.
Role of the M2-1 Transcription Antitermination Protein of Respiratory Syncytial Virus in Sequential Transcription
Authors: Fearns Rachel , Collins Peter L.
Published: December 2019

M2-1 protein of human respiratory syncytial virus (RSV) is a transcription antitermination factor that is important for the efficient synthesis of full-length mRNAs as well as for the synthesis of polycistronic readthrough mRNAs, which are characteristic of nonsegmented negative-strand RNA viruses. The contributions of these effects to RSV sequential transcription were investigated with minigenomes which contained one to five genes which were either foreign marker genes or authentic RSV genes. When evaluated on a promoter-proximal gene, the effect of M2-1 on the synthesis of full-length mRNA was much greater for a long (1,212- or 1,780-nucleotide) gene (up to a 615-fold increase) than for a short (274-nucleotide) gene (less than a 2-fold increase). This was independent of whether the gene contained non-RSV or RSV-specific sequence. Once the polymerase had terminated prematurely, it was unable to reinitiate at a downstream gene. These studies also confirmed that M2-1 enhances the synthesis of polycistronic mRNAs and that the magnitude of this effect varied greatly among different naturally occurring gene junctions. The synthesis of polycistronic mRNAs, which presumably involves antitermination at the gene-end signal, required a higher level of M2-1 than did the synthesis of the corresponding monocistronic mRNAs. M2-1 did not have a comparable antitermination effect at the junction between the leader region and the first gene. In a minigenome containing the NS1 and NS2 genes in their authentic sequence context, synthesis of full-length NS1 and NS2 mRNAs in the absence of M2-1 was remarkably high (36 and 57%, respectively, of the maximum levels observed in the presence of M2-1). In contrast, synthesis of mRNA from additional downstream genes was highly dependent on M2-1. Thus, RSV has the potential for two transcription programs: one in the absence of M2-1, in which only the NS1 and NS2 genes are transcribed, and one in the presence of M2-1, in which sequential transcription of the complete genome occurs. The dependence on M2-1 for transcription was greater for a gene in the fifth position from the promoter than for one in the third position. This indicates that under conditions where M2-1 is limiting, its concentration affects the gradient of transcription. Although M2-1 was found to have profound effects on transcription, it had no effect on replication of any minigenome tested, suggesting that it is not an active participant in RNA replication or regulation of RNA replication. Finally, since a permissive RSV infection is marked by a gradual increase in the intracellular accumulation of viral proteins including M2-1, we examined the relative abundances of various mRNAs during RSV infection for evidence of temporal regulation of transcription. None was found, implying that the availability of M2-1 during a permissive infection is sufficient at all times such that its concentration does not mediate temporal regulation of gene transcription.
Transient Association of Calnexin and Calreticulin with Newly Synthesized G1 and G2 Glycoproteins of Uukuniemi Virus (Family
Bunyaviridae
)
Authors: Veijola Johanna , Pettersson Ralf F.
Published: December 2019

The membrane glycoproteins G1 and G2 of Uukuniemi virus, a member of the
Bunyaviridae
family, are cotranslationally cleaved from a common precursor in the endoplasmic reticulum (ER). Here, we show that newly made G1 and G2 associate transiently with calnexin and calreticulin, two lectins involved in glycoprotein folding in the ER. Stable complexes between G1-G2 and calnexin or calreticulin could be immunoprecipitated after solubilization of virus-infected BHK21 cells with the detergents digitonin or Triton X-100. In addition, G1-G2-calnexin complexes could be recovered after solubilization with CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate}, while G1-G2-calreticulin complexes were not readily detected by using this detergent. Only endoglycosidase H-sensitive forms of G1 were found complexed with calnexin. Pulse-chase experiments showed that G1 and G2 associated with both chaperones transiently for up to 120 min. Sequential immunoprecipitations with anticalreticulin and anticalnexin antisera indicated that about 50% of newly synthesized G1 and G2 was associated with either calnexin or calreticulin. Our previous results have shown that newly synthesized G1 and G2 transiently interact also with the ER chaperone BiP and with protein disulfide isomerase (R. Persson and R. F. Pettersson, J. Cell Biol. 112:257–266, 1991). Taking all of this into consideration, we conclude that the folding of G1 and G2 in the ER is catalyzed by at least four different folding factors.
Identification of Human Herpesvirus 8-Specific Cytotoxic T-Cell Responses
Authors: Osman Mohamed , Kubo Toru , Gill Jasjit , Neipel Frank , Becker Marion , Smith Geoffrey , Weiss Robin , Gazzard Brian , Boshoff Chris , Gotch Frances
Published: December 2019

Human herpesvirus 8 (HHV-8) (or Kaposi’s sarcoma-associated herpesvirus) is implicated in the etiopathogenesis of Kaposi’s sarcoma (KS) and certain lymphoproliferations. The introduction of more effective therapies to treat human immunodeficiency virus infection has led to a decline in the incidence of KS and also in the resolution of KS in those already affected. This suggests that cellular immune responses including cytotoxic T lymphocytes (CTLs) could play a vital role in the control of HHV-8 infection and in KS pathogenesis. Here we elucidate HLA class I-restricted, HHV-8-specific cellular immune responses that could be important in the control of HHV-8 infection and subsequent tumor development. We show the presence of CTLs against HHV-8 latent (K12), lytic (K8.1), and highly variable (K1) proteins in infected individuals.
A Rhesus Macaque Rhadinovirus Related to Kaposi’s Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Encodes a Functional Homologue of Interleukin-6
Authors: Kaleeba Johnan A. R. , Bergquam Eric P. , Wong Scott W.
Published: December 2019

The rhesus rhadinovirus strain 17577 (RRV strain 17577) genome is essentially colinear with human herpesvirus 8 (HHV8)/Kaposi’s sarcoma-associated herpesvirus (KSHV) and encodes several analogous open reading frames (ORFs), including the homologue of cellular interleukin-6 (IL-6). To determine if the RRV IL-6-like ORF (RvIL-6) is biologically functional, it was expressed either transiently in COS-1 cells or purified from bacteria as a glutathioneS-transferase (GST)-RvIL-6 fusion and analyzed by IL-6 bioassays. Utilizing the IL-6-dependent B9 cell line, we found that both forms of RvIL-6 supported cell proliferation in a dose-dependent manner. Moreover, antibodies specific to the IL-6 receptor (IL-6R) or the gp130 subunit were capable of blocking the stimulatory effects of RvIL-6. Reciprocal titrations of GST-RvIL-6 against human recombinant IL-6 produced a more-than-additive stimulatory effect, suggesting that RvIL-6 does not inhibit but may instead potentiate normal cellular IL-6 signaling to B cells. These results demonstrate that RRV encodes an accessory protein with IL-6-like activity.
Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3C
pro
Proteinase
Authors: Martin Annette , Bénichou Danièle , Chao Shih-Fong , Cohen Lisette M. , Lemon Stanley M.
Published: December 2019

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family
Picornaviridae
, the primary cleavage of the HAV polyprotein is mediated by 3C
pro
, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3C
pro
, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3C
pro
proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3C
pro
that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3C
pro
recognition criteria and found that a substitution that ablates the presumed 3C
pro
dipeptide recognition sequence at Glu
764
-Ser
765
abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg
764
substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3C
pro
processing and may thus be uniquely dependent on a cellular proteinase.
Pathogenesis of Borna Disease Virus: Granulocyte Fractions of Psychiatric Patients Harbor Infectious Virus in the Absence of Antiviral Antibodies
Authors: Planz Oliver , Rentzsch Christine , Batra Anil , Batra Arvind , Winkler Tanja , Büttner Mathias , Rziha Hanns-Joachim , Stitz Lothar
Published: December 2019

Borna disease virus (BDV) causes acute and persistent infections in various vertebrates. During recent years, BDV-specific serum antibodies, BDV antigen, and BDV-specific nucleic acid were found in humans suffering from psychiatric disorders. Furthermore, viral antigen was detected in human autopsy brain tissue by immunohistochemical staining. Whether BDV infection can be associated with psychiatric disorders is still a matter of debate; no direct evidence has ever been presented. In the present study we report on (i) the detection of BDV-specific nucleic acid in human granulocyte cell fraction from three different psychiatric patients and (ii) the isolation of infectious BDV from these cells obtained from a patient with multiple psychiatric disorders. In leukocyte preparations other than granulocytes, either no BDV RNA was detected or positive PCR results were obtained only if there was at least 20% contamination with granulocytes. Parts of the antigenome of the isolated virus were sequenced, demonstrating the close relationship to the prototype BDV strains (He/80 and strain V) as well as to other human virus sequences. Our data provide strong evidence that cells in the granulocyte fraction represent the major if not the sole cell type harboring BDV-specific nucleic acid in human blood and contain infectious virus. In contrast to most other reports of putative human isolates, where sequences are virtually identical to those of the established laboratory strains, this isolate shows divergence in the region previously defined as variable in BDV from naturally infected animals.
Characterization of V3 Sequence Heterogeneity in Subtype C Human Immunodeficiency Virus Type 1 Isolates from Malawi: Underrepresentation of X4 Variants
Authors: Ping Li-Hua , Nelson Julie A. E. , Hoffman Irving F. , Schock Jody , Lamers Suzanna L. , Goodman Melissa , Vernazza Pietro , Kazembe Peter , Maida Martin , Zimba Dick , Goodenow Maureen M. , Eron Joseph J. , Fiscus Susan A. , Cohen Myron S. , Swanstrom Ronald
Published: December 2019

We have examined the nature of V3 sequence variability among subtype C human immunodeficiency virus type 1 (HIV-1) sequences from plasma-derived viral RNA present in infected men from Malawi. Sequence variability was assessed by direct sequence analysis of the V3 reverse transcription-PCR products, examination of virus populations by a subtype C V3-specific heteroduplex tracking assay (V3-HTA), and selected sequence analysis of molecular clones derived from the PCR products. Sequence variability in V3 among the subtype C viruses was not associated with the presence of basic amino acid substitutions. This observation is in contrast to that for subtype B HIV-1, where sequence variability is associated with such substitutions, and these substitutions are determinants of altered coreceptor usage. Evolutionary variants in subtype C V3 sequences, as defined by the V3-HTA, were not correlated with the CD4 level in the infected person, while such a correlation was found with subtype B V3 sequences. Viruses were isolated from a subset of the subjects; all isolates used CCR5 and not CXCR4 as a coreceptor, and none was able to grow in MT-2 cells, a hallmark of the syncytium-inducing phenotype that is correlated with CXCR4 usage. The overall sequence variability of the subtype C V3 region was no greater than that of the conserved regions of gp120. This limited sequence variability was also a feature of subtype B V3 sequences that do not carry the basic amino acid substitutions associated with altered coreceptor usage. Our results indicate that altered coreceptor usage is rare in subtype C HIV-1 isolates in sub-Saharan Africa and that sequence variability is not a feature of the V3 region ofenvin the absence of altered coreceptor usage.
The Gag Domains Required for Avian Retroviral RNA Encapsidation Determined by Using Two Independent Assays
Authors: Lee Eun-gyung , Yeo Ashly , Kraemer Brian , Wickens Marvin , Linial Maxine L.
Published: December 2019

The Rous sarcoma virus (RSV) Gag precursor polyprotein is the only viral protein which is necessary for specific packaging of genomic RNA. To map domains within Gag which are important for packaging, we constructed a series of Gag mutations in conjunction with a protease (PR) active-site point mutation in a full-length viral construct. We found that deletion of either the matrix (MA), the capsid (CA), or the protease (PR) domain did not abrogate packaging, although the MA domain is likely to be required for proper assembly. A previously characterized deletion of both Cys-His motifs in RSV nucleocapsid protein (NC) reduced both the efficiency of particle release and specific RNA packaging by 6- to 10-fold, consistent with previous observations that the NC Cys-His motifs played a role in assembly and RNA packaging. Most strikingly, when amino acid changes at Arg 549 and 551 immediately downstream of the distal NC Cys-His box were made, RNA packaging was reduced by more than 25-fold with no defect in particle release, demonstrating the importance of this basic amino acid region in packaging. We also used the yeast three-hybrid system to study avian retroviral RNA-Gag interactions. Using this assay, we found that the interactions of the minimal packaging region (Mψ) with Gag are of high affinity and specificity. Using a number of Mψ and Gag mutants, we have found a clear correlation between a reporter gene activation in a yeast three-hybrid binding system and an in vivo packaging assay. Our results showed that the binding assay provides a rapid genetic assay of both RNA and protein components for specific encapsidation.
Second-Site Reversion of a Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutant That Restores Enzyme Function and Replication Capacity
Authors: Olivares Isabel , Sánchez-Merino Víctor , Martínez Miguel A. , Domingo Esteban , López-Galíndez Cecilio , Menéndez-Arias Luis
Published: December 2019

Nonconservative substitutions for Tyr-115 in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) lead to enzymes displaying lower affinity for deoxynucleoside triphosphates (dNTPs) (A. M. Martı́n-Hernández, E. Domingo, and L. Menéndez-Arias, EMBO J. 15:4434–4442, 1996). Several mutations at this position (Y115W, Y115L, Y115A, and Y115D) were introduced in an infectious HIV-1 clone, and the replicative capacity of the mutant viruses was monitored. Y115W was the only mutant able to replicate in MT-4 cells, albeit very poorly. Nucleotide sequence analysis of the progeny virus recovered from supernatants of four independent transfection experiments showed that the Y115W mutation was maintained. However, in all cases an additional substitution in the primer grip of the RT (M230I) emerged when the virus increased its replication capacity. Using recombinant HIV-1 RT, we demonstrate that M230I mitigates the polymerase activity defect of the Y115W mutant, by increasing the dNTP binding affinity of the enzyme. The second-site suppressor effects observed were mediated by mutations in the 66-kDa subunit of the RT, as demonstrated with chimeric heterodimers. Examination of available crystal structures of HIV-1 RT suggests a possible mechanism for restoration of enzyme activity by the second-site revertant.
Changes in Rous Sarcoma Virus RNA Secondary Structure near the Primer Binding Site upon tRNA
Trp
Primer Annealing
Authors: Morris Shannon , Leis Jonathan
Published: December 2019

Predicted secondary-structure elements encompassing the primer binding site in the 5′ untranslated region of Rous sarcoma virus (RSV) RNA play an integral role in multiple viral replications steps including reverse transcription, DNA integration, and RNA packaging (A. Aiyar, D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis, J. Virol. 66:2464–2472, 1992; D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864–3872, 1991; J. T. Miller, Z. Ge, S. Morris, K. Das, and J. Leis, J. Virol. 71:7648–7656, 1997). These elements include the U5-Leader stem, U5-IR stem-loop, and U5-TΨC interaction region. Limited digestion of the 5′ untranslated region of wild-type and mutant RSV RNAs with structure- and/or sequence-specific RNases detects the presence of the U5-Leader stem and the U5-IR stem-loop. When a tRNA
Trp
primer is annealed to wild-type RNAs in vitro, limited nuclease mapping indicates that the U5-IR stem becomes partially unwound. This is not observed when mutant RNAs with altered U5-IR stem-loop structures are substituted for wild-type RNAs. The U5-Leader stem also becomes destabilized when the tRNA primer is annealed to either wild-type or mutant RNA fragments. Nuclease mapping studies of tRNA
Trp
, as well as the viral RNA, indicate that the U5-TΨC helix does form in vitro upon primer annealing. Collectively, these data suggest that the various structural elements near the RSV primer binding site undergo significant changes during the process of primer annealing.
Selective Irreversible Inactivation of Replicating Mengovirus by Nucleoside Analogues: a New Form of Viral Interference
Authors: Brdar Branko , Reich E.
Published: December 2019

We describe the selective irreversible inhibition of mengovirus growth in cultured cells by a combination of two pyrrolopyrimidine nucleoside analogues, 5-bromotubercidin (BrTu) and tubercidin (Tu). At a concentration of 5 μg/ml, BrTu reversibly blocked the synthesis of cellular mRNA and rRNA but did not inhibit either mengovirus RNA synthesis or multiplication. BrTu is a potent inhibitor of adenosine kinase, and low concentrations of BrTu (e.g., 0.5 μg/ml), which did not by themselves inhibit cell growth, blocked phosphorylation of Tu and thus protected uninfected cells against irreversible cytotoxicity resulting from Tu incorporation into nucleic acids. In contrast, in mengovirus-infected cells, BrTu did not completely inhibit Tu incorporation into mengovirus RNA, allowing the formation of Tu-containing functionally defective polynucleotides that aborted the virus development cycle. This increased incorporation of Tu coupled to mengovirus infection could be attributed either to a reduction in the inhibitory action of BrTu and/or its nucleotide derivatives at the level of nucleoside and nucleotide kinases and/or, perhaps, to an effect upon the nucleoside transport system. The virus life cycle in nucleoside-treated cells progressed to the point of synthesis of negative strands and probably to the production of a few defective new positive strands. Irreversible virus growth arrest was achieved if the nucleoside mixture of BrTu (0.5 to 10 μg/ml) and Tu (1 to 20 μg/ml) was added no later than 30 min after virus infection and maintained for periods of 2 to 8 h. The cultures thus “cured” of mengovirus infection could be maintained and transferred for several weeks, during which they neither produced detectable virus nor showed a visible cytopathic effect; however, the infected and cured cells themselves, while metabolically viable, were permanently impaired in RNA synthesis and unable to divide. Although completely resistant to superinfecting picornaviruses, they retained the ability to support the growth of several other viruses (vaccinia virus, reovirus, and vesicular stomatitis virus), showing that cured cells had, in general, retained the metabolic and structural machinery needed for virus production. The resistance of cured cells to superinfection with picornaviruses seemed attributable neither to interferon action nor to destruction or blockade of virus receptors but more likely to the consumption of some host factor(s) involved in the expression of early viral functions during the original infection.
RNase H Requirements for the Second Strand Transfer Reaction of Human Immunodeficiency Virus Type 1 Reverse Transcription
Authors: Smith Christine M. , Smith Jeffrey S. , Roth Monica J.
Published: December 2019

Retroviral reverse transcriptase (RT) enzymes are responsible for transcribing viral RNA into double-stranded DNA. An in vitro assay to analyze the second strand transfer event during human immunodeficiency virus type 1 (HIV-1) reverse transcription has been developed. Model substrates were constructed which mimic the viral intermediate found during plus-strand strong-stop synthesis. Utilizing wild-type HIV-1 RT and a mutant E478Q RT, the requirement for RNase H activity in this strand transfer event was analyzed. In the presence of Mg
2+
, HIV-1 RT was able to fully support the second strand transfer reaction in vitro. However, in the presence of Mg
2+
, the E478Q RT mutant had no detectable RNase H activity and was unable to support strand transfer. In the presence of Mn
2+
, the E478Q RT yields the initial endoribonucleolytic cleavage at the penultimate C residue of the tRNA primer yet does not support strand transfer. This suggests that subsequent degradation of the RNA primer by the RNase H domain was required for strand transfer. In reactions in which the E478Q RT was complemented with exogenous RNase H enzymes, strand transfer was supported. Additionally, we have shown that HIV-1 RT is capable of supporting strand transfer with substrates that mimic tRNA
His
as well as the authentic tRNA
3
Lys
.
Escape of Human Cytomegalovirus from HLA-DR-Restricted CD4+T-Cell Response Is Mediated by Repression of Gamma Interferon-Induced Class II Transactivator Expression
Authors: Le Roy Emmanuelle , Mühlethaler-Mottet Annick , Davrinche Christian , Mach Bernard , Davignon Jean-Luc
Published: December 2019

Human cytomegalovirus (HCMV), a betaherpesvirus, is a pathogen which escapes immune recognition through various mechanisms. In this paper, we show that HCMV down regulates gamma interferon (IFN-γ)-induced HLA-DR expression in U373 MG astrocytoma cells due to a defect downstream of STAT1 phosphorylation and nuclear translocation. Repression of class II transactivator (CIITA) mRNA expression is detected within the first hours of IFN-γ–HCMV coincubation and results in the absence of HLA-DR synthesis. This defect leads to the absence of presentation of the major immediate-early protein IE1 to specific CD4+T-cell clones when U373 MG cells, used as antigen-presenting cells, are treated with IFN-γ plus HCMV. However, presentation of endogenously synthesized IE1 can be restored when U373 MG cells are transfected with CIITA prior to infection with HCMV. Altogether, the data indicate that the defect induced by HCMV resides in the activation of the IFN-γ-responsive promoter of CIITA. This is the first demonstration of a viral inhibition of CIITA expression.
Modified VP22 Localizes to the Cell Nucleus during Synchronized Herpes Simplex Virus Type 1 Infection
Authors: Pomeranz Lisa E. , Blaho John A.
Published: December 2019

The U
L
49 gene product (VP22) of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is a virion phosphoprotein which accumulates inside infected cells at late stages of infection. We previously (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401–17410, 1994) discovered that the form of VP22 packaged into infectious virions differed from VP22 extracted from infected-cell nuclei in that the virion-associated form had a higher electrophoretic mobility in denaturing gels. Based on these results, we proposed that VP22 in virions was “undermodified” in some way. The goal of this study is to document the biological and biochemical properties of VP22 throughout the entire course of a productive HSV-1 infection. We now report the following. (i) VP22 found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear, as measured by indirect immunofluorescence. (ii) Using a synchronized infection system, we determined that VP22 exists predominantly in the cytoplasm early in infection and accumulates in the nucleus late in infection. (iii) While cytoplasmic VP22 colocalizes with the HSV-1 glycoprotein D early in infection, the nuclear form of VP22 is not restricted to replication compartments which accumulate ICP4. (iv) VP22 migrates as at least three unique electrophoretic species in denaturing sodium dodecyl sulfate-DATD-polyacrylamide gels. VP22a, VP22b, and VP22c have high, intermediate, and low mobility, respectively. (v) The relative distribution of the various forms of VP22 derived from infected whole-cell extracts varies during the course of infection such that low-mobility species predominate at early times and high-mobility forms accumulate later. (vi) The highest-mobility forms of VP22 partition with the cytoplasmic fraction of infected cells, while the lowest-mobility forms are associated with the nuclear fraction. (vii) Finally, full-length VP22 which partitions in the nucleus incorporates radiolabel from [
32
P]orthophosphate whereas cytoplasmic VP22 does not. Based on these results, we conclude that modification of VP22 coincides with its appearance in the nucleus during the course of productive HSV-1 infection.
Phosphorylation and/or Presence of Serine 37 in the Movement Protein of Tomato Mosaic Tobamovirus Is Essential for Intracellular Localization and Stability In Vivo
Authors: Kawakami Shigeki , Padgett Hal S. , Hosokawa Daijiro , Okada Yoshimi , Beachy Roger N. , Watanabe Yuichiro
Published: December 2019

The P30 movement protein (MP) of tomato mosaic tobamovirus (ToMV) is synthesized in the early stages of infection and is phosphorylated in vivo. Here, we determined that serine 37 and serine 238 in the ToMV MP are sites of phosphorylation. MP mutants in which serine was replaced by alanine at positions 37 and 238 (LQ37A238A) or at position 37 only (LQ37A) were not phosphorylated, and mutant viruses did not infect tobacco or tomato plants. By contrast, mutation of serine 238 to alanine did not affect the infectivity of the virus (LQ238A). To investigate the subcellular localization of mutant MPs, we constructed viruses that expressed each mutant MP fused with the green fluorescent protein (GFP) of
Aequorea victoria
. Wild-type and mutant LQ238A MP fusion proteins showed distinct temporally regulated patterns of MP-GFP localization in protoplasts and formation of fluorescent ring-shaped infection sites on
Nicotiana benthamiana
. However mutant virus LQ37A MP-GFP did not show a distinct pattern of localization or formation of fluorescent rings. Pulse-chase experiments revealed that MP produced by mutant virus LQ37A was less stable than wild-type and LQ238A MPs. MP which contained threonine at position 37 was phosphorylated, but the stability of the MP in vivo was very low. These studies suggest that the presence of serine at position 37 or phosphorylation of serine 37 is essential for intracellular localization and stability of the MP, which is necessary for the protein to function.
Neural Stem Cells as Engraftable Packaging Lines Can Mediate Gene Delivery to Microglia: Evidence from Studying Retroviral
env
-Related Neurodegeneration
Authors: Lynch William P. , Sharpe Arlene H. , Snyder Evan Y.
Published: December 2019

The induction of spongiform myeloencephalopathy by murine leukemia viruses is mediated primarily by infection of central nervous system (CNS) microglia. In this regard, we have previously shown that CasBrE-induced disease requires late, rather than early, virus replication events in microglial cells (W. P. Lynch et al., J. Virol. 70:8896–8907, 1996). Furthermore, neurodegeneration requires the presence of unique sequences within the viral
env
gene. Thus, the neurodegeneration-inducing events could result from microglial expression of retroviral envelope protein alone or from the interaction of envelope protein with other viral structural proteins in the virus assembly and maturation process. To distinguish between these possible mechanisms of disease induction, we engineered the engraftable neural stem cell line C17-2 into packaging/producer cells in order to deliver the neurovirulent CasBrE
env
gene to endogenous CNS cells. This strategy resulted in significant CasBrE
env
expression within CNS microglia without the appearance of replication competent virus. CasBrE envelope expression within microglia was accompanied by increased expression of activation markers F4/80 and Mac-1 (CD11b) but failed to induce spongiform neurodegenerative changes. These results suggest that envelope expression alone within microglia is not sufficient to induce neurodegeneration. Rather, microglia-mediated disease appears to require neurovirulent Env protein interaction with other viral proteins during assembly or maturation. More broadly, the results presented here prove the efficacy of a novel method by which neural stem cell biology may be harnessed for genetically manipulating the CNS, not only for studying neurodegeneration but also as a paradigm for the disseminated distribution of retroviral vector-transduced genes.
Association with the Cellular Export Receptor CRM 1 Mediates Function and Intracellular Localization of Epstein-Barr Virus SM Protein, a Regulator of Gene Expression
Authors: Boyle Sarah M. , Ruvolo Vivian , Gupta Ashish K. , Swaminathan Sankar
Published: December 2019

Splicing and posttranscriptional processing of eukaryotic gene transcripts are linked to their nuclear export and cytoplasmic expression. Unspliced pre-mRNAs and intronless transcripts are thus inherently poorly expressed. Nevertheless, human and animal viruses encode essential genes as single open reading frames or in the intervening sequences of other genes. Many retroviruses have evolved mechanisms to facilitate nuclear export of their unspliced mRNAs. For example, the human immunodeficiency virus RNA-binding protein Rev associates with the soluble cellular export receptor CRM 1 (exportin 1), which mediates nucleocytoplasmic translocation of Rev-HIV RNA complexes through the nuclear pore. The transforming human herpesvirus Epstein-Barr virus (EBV) expresses a nuclear protein, SM, early in its lytic cycle; SM binds RNA and posttranscriptionally activates expression of certain intronless lytic EBV genes. Here we show that both the
trans
-activation function and cytoplasmic translocation of SM are dependent on association with CRM 1 in vivo. SM is also shown to be associated in vivo with other components of the CRM 1 export pathway, including the small GTPase Ran and the nucleoporin CAN/Nup214. SM is shown to be present in the cytoplasm, nucleoplasm, and nuclear envelope of transfected cells. Mutation of a leucine-rich region (LRR) of SM inhibited CRM 1-mediated cytoplasmic translocation and SM activity, as did leptomycin B, an inhibitor of CRM 1 complex formation. Surprisingly, however, leptomycin B treatment and mutation of the LRR both led to SM becoming more tightly attached to intranuclear structures. These findings suggest a model in which SM is not merely a soluble carrier protein for RNA but rather is bound directly to intranuclear proteins, possibly including the nuclear pore complex.
Retrovirus Targeting by Tropism Restriction to Melanoma Cells
Authors: Martin F. , Neil S. , Kupsch J. , Maurice M. , Cosset F.-L. , Collins M.
Published: December 2019

Targeted vectors will be necessary for many gene therapy applications. To target retroviruses to melanomas, we fused a single-chain variable fragment antibody (scFv) directed against the surface glycoprotein high-molecular-weight melanoma-associated antigen (HMW-MAA) to the amphotropic murine leukemia virus envelope. A proline-rich hinge and matrix metalloprotease (MMP) cleavage site linked the two proteins. The modified viruses bound only to HMW-MAA-expressing cells, as inclusion of the proline-rich hinge prevented viral binding to the amphotropic viral receptor. Following attachment to HMW-MAA, MMP cleavage of the envelope at the melanoma cell surface removed the scFv and proline-rich hinge, allowing infection. Complexing of targeted retroviruses with 2,3-dioleoyloxy-
N
-[2(spermine-carboxamido)ethyl]
N
,
N
-dimethyl-1-propanaminium trifluoroacetate–dioleoyl phosphatidylethanolamine liposomes greatly increased their efficiency without affecting their target cell specificity. In a cell mixture, 40% of HMW-MAA-positive cells but less than 0.01% of HMW-MAA-negative cells were infected. This approach can therefore produce efficient, targeted retroviruses suitable for in vivo gene delivery and should allow specific gene delivery to many human cell types by inclusion of different scFv and protease combinations.
VP1, the Putative RNA-Dependent RNA Polymerase of Infectious Bursal Disease Virus, Forms Complexes with the Capsid Protein VP3, Leading to Efficient Encapsidation into Virus-Like Particles
Authors: Lombardo Eleuterio , Maraver Antonio , Castón José R. , Rivera José , Fernández-Arias Armando , Serrano Antonio , Carrascosa José L. , Rodriguez José F.
Published: December 2019

A cDNA corresponding to the coding region of VP1, the putative RNA-dependent RNA polymerase, of infectious bursal disease virus (IBDV) was cloned and inserted into the genome of a vaccinia virus inducible expression vector. The molecular mass and antigenic reactivity of VP1 expressed in mammalian cells are identical to those of its counterpart expressed in IBDV-infected cells. The results presented here demonstrate that VP1 is efficiently incorporated into IBDV virus-like particles (VLPs) produced in mammalian cells coexpressing the IBDV polyprotein and VP1. Incorporation of VP1 into VLPs requires neither the presence of IBDV RNAs nor that of the nonstructural polypeptide VP5. Immunofluorescence, confocal laser scanning microscopy, and immunoprecipitation analyses conclusively showed that VP1 forms complexes with the structural polypeptide VP3. Formation of VP1-VP3 complexes is likely to be a key step for the morphogenesis of IBDV particles.
The Presence of Human Immunodeficiency Virus Type 1 Vpr Correlates with a Decrease in the Frequency of Mutations in a Plasmid Shuttle Vector
Authors: Jowett Jeremy B. , Xie Yi-ming , Chen Irvin S. Y.
Published: December 2019

The human immunodeficiency virus type 1 (HIV-1) Vpr protein induces cell cycle arrest at the border of G
2
and M similar to the arrest caused by agents which damage DNA. We determined whether the presence of Vpr would affect the ability of cells to repair DNA. We developed a shuttle vector system to analyze the effect of Vpr upon the repair of UV-damaged DNA. Our results demonstrated that the presence of Vpr decreased the rate of deletions in this system. Of note, cells arrested in G
2
by other genotoxic agents also increased the frequency of DNA repair of UV-damaged shuttle vectors. We did not observe any direct effect of Vpr upon the rate of double-strand break repair and/or nucleotide excision repair of genomic DNA in cells. Our results suggest a role for HIV-1 Vpr in altering the frequency of DNA repair, a property which may have importance for HIV-1 replication and pathogenesis.
An Endoplasmic Reticulum Retrieval Signal Partitions Human Foamy Virus Maturation to Intracytoplasmic Membranes
Authors: Goepfert Paul A. , Shaw Kit , Wang George , Bansal Anju , Edwards Bradley H. , Mulligan Mark J.
Published: December 2019

Among all retroviruses, foamy viruses (FVs) are unique in that they regularly mature at intracytoplasmic membranes. The envelope glycoprotein of FV encodes an endoplasmic reticulum (ER) retrieval signal, the dilysine motif (KKXX), that functions to localize the human FV (HFV) glycoprotein to the ER. This study analyzed the function of the dilysine motif in the context of infectious molecular clones of HFV that encoded mutations in the dilysine motif. Electron microscopy (EM) demonstrated virion budding both intracytoplasmically and at the plasma membrane for the wild-type and mutant viruses. Additionally, mutant viruses retained their infectivity, but viruses lacking the dilysine signal budded at the plasma membrane to a greater extent than did wild-type viruses. Interestingly, this relative increase in budding across the plasma membrane did not increase the overall release of viral particles into cell culture media as measured by protein levels in viral pellets or infectious virus titers. We conclude that the dilysine motif of HFV imposes a partial restriction on the site of viral maturation but is not necessary for viral infectivity.
Deletion of the R78 G Protein-Coupled Receptor Gene from Rat Cytomegalovirus Results in an Attenuated, Syncytium-Inducing Mutant Strain
Authors: Beisser Patrick S. , Grauls Gert , Bruggeman Cathrien A. , Vink Cornelis
Published: December 2019

The rat cytomegalovirus (RCMV) R78 gene belongs to an uncharacterized class of viral G protein-coupled receptor (GCR) genes. The predicted amino acid sequence of the R78 open reading frame (ORF) shows 25 and 20% similarity with the gene products of murine cytomegalovirus M78 and human cytomegalovirus UL78, respectively. The R78 gene is transcribed throughout the early and late phases of infection in rat embryo fibroblasts (REF) in vitro. Transcription of R78 was found to result in three different mRNAs: (i) a 1.8-kb mRNA containing the R78 sequence, (ii) a 3.7-kb mRNA containing both R77 and R78 sequences, and (iii) a 5.7-kb mRNA containing at least ORF R77 and ORF R78 sequences. To investigate the function of the R78 gene, we generated two different recombinant virus strains: an RCMV R78 null mutant (RCMVΔR78a) and an RCMV mutant encoding a GCR from which the putative intracellular C terminus has been deleted (RCMVΔR78c). These recombinant viruses replicated with a 10- to 100-fold-lower efficiency than wild-type (wt) virus in vitro. Interestingly, unlike wt virus-infected REF, REF infected with the recombinants develop a syncytium-like appearance. A striking difference between wt and recombinant viruses was also seen in vivo: a considerably higher survival was seen among recombinant virus-infected rats than among RCMV-infected rats. We conclude that the RCMV R78 gene encodes a novel GCR-like polypeptide that plays an important role in both RCMV replication in vitro and the pathogenesis of viral infection in vivo.
Hepatitis B Virus X Protein Is both a Substrate and a Potential Inhibitor of the Proteasome Complex
Authors: Hu Zongyi , Zhang Zhensheng , Doo Edward , Coux Olivier , Goldberg Alfred L. , Liang T. Jake
Published: December 2019

The hepatitis B virus X protein (HBX) is essential for the establishment of HBV infection in vivo and exerts a pleiotropic effect on diverse cellular functions. The yeast two-hybrid system had indicated that HBX could interact with two subunits of the 26S proteasome. Here we demonstrate an association in vivo of HBX with the 26S proteasome complex by coimmunoprecipitation and colocalization upon sucrose gradient centrifugation. Expression of HBX in HepG2 cells caused a modest decrease in the proteasome’s chymotrypsin- and trypsin-like activities and in hydrolysis of ubiquitinated lysozyme, suggesting that HBX functions as an inhibitor of proteasome. In these cells, HBX is degraded with a half-life of 30 min. Proteasome inhibitors retarded this rapid degradation and caused a marked increase in the level of HBX and an accumulation of HBX in polyubiquitinated form. Thus, the low intracellular level of HBX is due to rapid proteolysis by the ubiquitin-proteasome pathway. Surprisingly, the proteasome inhibitors blocked the transactivation by HBX, and this effect was not a result of a squelching phenomenon due to HBX accumulation. Therefore, proteasome function is possibly required for the transactivation function of HBX. The inhibition of protein breakdown by proteasomes may account for the multiple actions of HBX and may be an important feature of HBV infection, possibly in helping stabilize viral gene products and suppressing antigen presentation.
Temperature-Sensitive Lesions in Two Influenza A Viruses Defective for Replicative Transcription Disrupt RNA Binding by the Nucleoprotein
Authors: Medcalf Liz , Poole Emma , Elton Debra , Digard Paul
Published: December 2019

The negative-sense segmented RNA genome of influenza virus is transcribed into capped and polyadenylated mRNAs, as well as full-length replicative intermediates (cRNAs). The mechanism that regulates the two forms of transcription remains unclear, although several lines of evidence imply a role for the viral nucleoprotein (NP). In particular, temperature-shift and biochemical analyses of the temperature-sensitive viruses A/WSN/33
ts
56 and A/FPV/Rostock/34/Giessen
ts
G81 containing point mutations within the NP coding region have indicated specific defects in replicative transcription at the nonpermissive temperature. To identify the functional defect, we introduced the relevant mutations into the NP of influenza virus strain A/PR/8/34. Both mutants were temperature sensitive for influenza virus gene expression in transient-transfection experiments but localized and accumulated normally in transfected cells. Similarly, the mutants retained the ability to self-associate and interact with the virus polymerase complex whether synthesized at the permissive or the nonpermissive temperatures. In contrast, the mutant NPs were defective for RNA binding when expressed at the nonpermissive temperature but not when expressed at 30°C. This suggests that the RNA-binding activity of NP is required for replicative transcription.
High-Fidelity Correction of Mutations at Multiple Chromosomal Positions by Adeno-Associated Virus Vectors
Authors: Inoue Naoki , Hirata Roli K. , Russell David W.
Published: December 2019

The gene targeting techniques used to modify chromosomes in mouse embryonic stem cells have had limited success with many other cell types, especially normal primary cells with restricted growth capacity outside the organism. This is due in large part to the technical problems and/or inefficiency of conventional DNA transfer methods, as well as the low rates of homologous recombination obtained in unselected cell populations. We recently described an alternative approach in which adeno-associated virus (AAV) vectors were used to modify homologous chromosomal sequences, and targeting rates close to 1% were observed at the single copy hypoxanthine phosphoribosyl transferase (HPRT) locus in normal human cells (D. W. Russell and R. K. Hirata, Nat. Genet. 18:325–330, 1998). Here we report experiments in which we used a retroviral shuttle vector system to introduce and characterize target loci in human chromosomes, and demonstrate that AAV vectors can correct several types of mutations with high fidelity, independent of chromosomal position. The gene targeting rates varied depending on the type of mutation being corrected, implicating cellular mismatch recognition functions in the reaction. Since AAV vectors can efficiently deliver DNA to many cell types both in vivo and ex vivo, our results suggest that AAV-mediated gene targeting will have wide applicability, including therapeutic gene correction.
DNA Unwinding Functions of Minute Virus of Mice NS1 Protein Are Modulated Specifically by the Lambda Isoform of Protein Kinase C
Authors: Dettwiler Sabine , Rommelaere Jean , Nüesch Jürg P. F.
Published: December 2019

The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966–9977, 1998). The present study led to the identification of atypical PKCλ/ι as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKCλ phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKCλ phosphorylation at residue S473.