bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Immunodeficiency Virus Type 1 Integrase Protein Promotes Reverse Transcription through Specific Interactions with the Nucleoprotein Reverse Transcription Complex
Authors: Wu Xiaoyun , Liu Hongmei , Xiao Hongling , Conway Joan A. , Hehl Eric , Kalpana Ganjam V. , Prasad Vinayaka , Kappes John C.
Published: December 2019

The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN) is essential for integration of the viral DNA into host cell chromosomes. Since IN is expressed and assembled into virions as part of the 160-kDa Gag-Pol precursor polyprotein and catalyzes integration of the provirus in infected cells as a mature 32-kDa protein, mutations in IN are pleiotropic and may affect virus replication at different stages of the virus life cycle in addition to integration. Several different phenotypes have been observed for IN mutant viruses, including defects in virion morphology, protein composition, reverse transcription, nuclear import, and integration. Because the effects of mutations in the IN domain of Gag-Pol can not always be distinguished from those of mutations in the mature IN protein, there remains a significant gap in our understanding of IN function in vivo. To directly analyze the function of the mature IN protein itself, in the context of a replicating virus but independently from that of Gag-Pol, we used an approach developed in our laboratory for incorporating proteins into HIV virions by their expression in
trans
as fusion partners of either Vpr or Vpx. By providing IN in
trans
as a Vpr-IN fusion protein, our analysis revealed, for the first time, that the mature IN protein is essential for the efficient initiation of reverse transcription in infected cells and that this function does not require the IN protein to be enzymatically (integration) active. Our findings of a direct physical interaction between IN and reverse transcriptase and the failure of heterologous HIV-2 IN protein to efficiently support reverse transcription indicate that this novel function occurs through specific interactions with other viral components of the reverse transcription initiation complex. Studies involving complementation between integration- and DNA synthesis-defective IN mutants further support this conclusion and reveal that the highly conserved HHCC motif of IN is important for both activities. These findings provide important new insights into IN function and reverse transcription in the context of the nucleoprotein reverse transcription complex within the infected cell. Moreover, they validate a novel approach that obviates the need to mutate Gag-Pol in order to study the role of its individual mature components at the virus replication level.
Suppressor Mutations within the Core Binding Factor (CBF/AML1) Binding Site of a T-Cell Lymphomagenic Retrovirus
Authors: Martiney Marita J. , Levy Laura S. , Lenz Jack
Published: December 2019

The transcriptional enhancer of the lymphomagenic mouse retrovirus SL3 contains a binding site for the transcription factor core binding factor (CBF; also called AML1, PEBP2, and SEF1). The SL3 CBF binding site is called the core. It differs from the core of the weakly lymphomagenic mouse retrovirus Akv by one nucleotide (the sequences are TGTGGTTAA and TGTGGTCAA, respectively). A mutant virus called SAA that was identical to SL3 except that its core was mutated to the Akv sequence was only moderately attenuated for lymphomagenicity. In most SAA-infected mice, tumor proviruses contained either reversions of the original mutation or one of two novel core sequences. In 20% of the SAA-infected mice, tumor proviruses retained the original SAA/Akv core mutation but acquired one of two additional mutations (underlined), TG
C
GGTCAA or TGTGGTC
T
A, that generated core elements called So and T*, respectively. We tested whether the novel base changes in the So and T* cores were suppressor mutations. SL3 mutants that contained So or T* cores in place of the wild-type sequence were generated. These viruses induced T-cell lymphomas in mice more quickly than SAA. Therefore, the mutations in the So and T* cores are indeed second-site suppressor mutations. The suppressor mutations increased CBF binding in vitro and transcriptional activity of the viral long terminal repeats (LTRs) in T lymphocytes to levels comparable to those of SL3. Thus, CBF binding was increased by any of three different nucleotide changes within the sequence of the SAA core. Increased CBF binding resulted in increased LTR transcriptional activity in T cells and in increased viral lymphomagenicity.
Identification of an Active Reverse Transcriptase Enzyme Encoded by a Human Endogenous HERV-K Retrovirus
Authors: Berkhout Ben , Jebbink Maarten , Zsíros Jozsef
Published: December 2019

Of the numerous endogenous retroviral elements that are present in the human genome, the abundant HERV-K family is distinct because several members are transcriptionally active and coding for biologically active proteins. A detailed phylogeny of the HERV-K family based on the partial sequence of the reverse transcriptase (RT) gene revealed a high incidence of an intact RT open reading frame within the HML-2 subgroup of HERV-K elements. In this study, we report the cloning of six full-length HML-2 RT genes, of which five contain an uninterrupted open reading frame. The RT enzymes were expressed as glutathione
S
-transferase fusion proteins in
Escherichia coli
, and several HERV-K RT enzymes demonstrated polymerase as well as RNase H activity. Several biochemical properties of the RT polymerase were analyzed, including the template requirements and optimal reaction conditions (temperature, type of divalent cation). Inspection of the nucleotide sequence of the HERV-K RT genes demonstrated a mosaic structure, suggesting that a high level of genetic recombination has occurred in this virus family, which is a hallmark of replication by means of reverse transcription. The selective pressure to maintain the RT coding potential is illustrated by the sequence of a particular HERV-K isolate that contains three 1-nucleotide deletions within a small RT segment, thus maintaining the open reading frame. These combined results may suggest that these endogenous RT enzymes still have a biological function. It is possible that the RT activity was involved in the spread of this major class of retroelements by retrotransposition, and in fact it cannot be excluded that this retrovirus group is still mobile. The endogenous RT activity may also have been involved in the shaping of the human genome, e.g., by formation of pseudogenes.
Interaction between the Negative Regulator of Splicing Element and a 3′ Splice Site: Requirement for U1 Small Nuclear Ribonucleoprotein and the 3′ Splice Site Branch Point/Pyrimidine Tract
Authors: Cook Craig R. , McNally Mark T.
Published: December 2019

The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several
cis
elements that account for the accumulation of large amounts of unspliced RNA for use as
gag-pol
mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5′ splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3′ splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3′ splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3′ splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3′ splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3′ splice site and suggest that U1 is of primary importance for NRS splicing inhibition.
In Vivo Addition of Poly(A) Tail and AU-Rich Sequences to the 3′ Terminus of the Sindbis Virus RNA Genome: a Novel 3′-End Repair Pathway
Authors: Raju Ramaswamy , Hajjou Mustapha , Hill Kristie R. , Botta Vandana , Botta Sisir
Published: December 2019

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5′ end and polyadenylated at its 3′ end. The 3′ nontranslated region (3′NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3′CSE) and a poly(A) tail. This 3′CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693–2704, 1997). Here, we demonstrate that the 3′-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3′NTR, we generated a battery of mutant genomes with mutations in the 3′NTR and tested their ability to generate infectious virus and undergo 3′ polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3′CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3′CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3′NTR. These results indicate the ability of alphavirus RNAs to undergo 3′ repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3′ end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.
Interclass Transmission and Phyletic Host Tracking in Murine Leukemia Virus-Related Retroviruses
Authors: Martin Joanne , Herniou Elisabeth , Cook James , O’Neill Rachel Waugh , Tristem Michael
Published: December 2019

Retroviruses are capable of infectious horizontal transmission between hosts, usually between individuals within a single species, although a number of probable zoonotic infections resulting from transmission between different species of placental mammals have also been reported. Despite these data, it remains unclear how often interspecies transmission events occur or whether their frequency is influenced by the evolutionary distance between host taxa. To address this problem we used PCR to amplify and characterize endogenous retroviruses related to the murine leukemia viruses. We show that members of this retroviral genus are harbored by considerably more organisms than previously thought and that phylogenetic analysis demonstrates that viruses isolated from a particular host class generally cluster together, suggesting that infectious virus horizontal transfer between vertebrate classes occurs only rarely. However, two recent instances of transmission of zoonotic infections between distantly related host organisms were identified. One, from mammals to birds, has led to a rapid adaptive radiation into other avian hosts. The other, between placental and marsupial mammals, involves viruses clustering with recently described porcine retroviruses, adding to concerns regarding the xenotransplantation of pig organs to humans.
A Role for Perforin in Downregulating T-Cell Responses during Chronic Viral Infection
Authors: Matloubian Mehrdad , Suresh M. , Glass Alison , Galvan Marisa , Chow Kit , Whitmire Jason K. , Walsh Craig M. , Clark William R. , Ahmed Rafi
Published: December 2019

Cytotoxic T cells secrete perforin to kill virus-infected cells. In this study we show that perforin also plays a role in immune regulation. Perforin-deficient (perf −/−) mice chronically infected with lymphocytic choriomeningitis virus (LCMV) contained greater numbers of antiviral T cells compared to persistently infected +/+ mice. The enhanced expansion was seen in both CD4 and CD8 T cells, but the most striking difference was in the numbers of LCMV-specific CD8 T cells present in infected perf −/− mice. Persistent LCMV infection of +/+ mice results in both deletion and anergy of antigen-specific CD8 T cells, and our results show that this peripheral “exhaustion” of activated CD8 T cells occurred less efficiently in perf −/− mice. This excessive accumulation of activated CD8 T cells resulted in immune-mediated damage in persistently infected perf −/− mice; ∼50% of these mice died within 2 to 4 weeks, and mortality was fully reversed by in vivo depletion of CD8 T cells. This finding highlights an interesting dichotomy between the role of perforin in viral clearance and immunopathology; perforin-deficient CD8 T cells were unable to clear the LCMV infection but were capable of causing immune-mediated damage. Finally, this study shows that perforin also plays a role in regulating T-cell-mediated autoimmunity. Mice that were deficient in both perforin and Fas exhibited a striking acceleration of the spontaneous lymphoproliferative disease seen in Fas-deficient (lpr) mice. Taken together, these results show that the perforin-mediated pathway is involved in downregulating T-cell responses during chronic viral infection and autoimmunity and that perforin and Fas act independently as negative regulators of activated T cells.
Group D Adenoviruses Infect Primary Central Nervous System Cells More Efficiently than Those from Group C
Authors: Chillon Miguel , Bosch Assumpció , Zabner Joseph , Law Lane , Armentano Donna , Welsh Michael J. , Davidson Beverly L.
Published: December 2019

Group C adenovirus-mediated gene transfer to central nervous system cells is inefficient. We found that wild-type group D viruses, or recombinant adenovirus type 2 (Ad2) (group C) modified to contain Ad17 (group D) fiber, were more efficient in infecting primary cultures of neurons. Together with studies on primary vascular endothelial cells and tissue culture cell lines, our results indicate that there is not a universally applicable adenovirus serotype for use as a gene transfer vector.
PrM- and Cell-Binding Domains of the Dengue Virus E Protein
Authors: Wang Songli , He Runtao , Anderson Robert
Published: December 2019

The E-prM proteins of flaviviruses are unusual complexes which play important roles in virus assembly and fusion modulation and in potential immunity-inducing vaccines. Despite their importance, little is known about the biogenesis and structural organization of E-prM complexes. Pulse-chase radiolabeling of dengue virus-infected Vero cells demonstrated a rapid interassociation of E and prM proteins, and sucrose gradient sedimentation analysis suggested that E-prM complexes progressed from simple heteromers to more densely sedimenting structures indicating increased multimerization. E-prM heteromers of even higher complexity were observed in virus particles, suggesting an intracellular assembly process which results in the networking of E-prM subunits into a lattice-like structure found in virus particles. Trypsin cleavage of E-prM-containing virus particles resulted in the release of a soluble 45-kDa fragment of the E protein which retained cell-binding activity. The results suggest that E-prM interactions in dengue virus particles are largely mediated by domains in the carboxy-terminal anchoring domain of E, while cell-binding activity is retained in a trypsin-releasable ectodomain of the E protein.
Neoplastic Transformation-Associated Stimulation of the In Vitro Resolution of Concatemer Junction Fragments from Minute Virus of Mice DNA
Authors: Kuntz-Simon Gaëlle , Bashir Tarig , Rommelaere Jean , Willwand Kurt
Published: December 2019

Minute virus of mice (MVM) shows an oncotropic behavior reflected by its ability to amplify its genome more efficiently in a number of transformed versus normal cells. In vivo and in vitro studies revealed that the major effect of cell transformation on MVM DNA replication occurs at the level of double-stranded replicative-form amplification. In particular, resolution of MVM DNA concatemers into monomers was found to be highly sensitive to neoplastic transformation.
Resistance of Interleukin-1β-Deficient Mice to Fatal Sindbis Virus Encephalitis
Authors: Liang Xiao Huan , Goldman James E. , Jiang Hui Hui , Levine Beth
Published: December 2019

Interleukin-1β (IL-1β) concentrations are frequently elevated in central nervous system (CNS) viral infections, but the pathophysiologic significance of such elevations is not known. To examine the role of IL-1β in CNS viral pathogenesis, we compared the natural histories of IL-1β-deficient and wild-type 129 SV(ev) mice infected with a neurovirulent viral strain, neuroadapted Sindbis virus (NSV). We found that the incidence of severe paralysis and death was markedly decreased in NSV-infected IL-1β−/−mice compared to NSV-infected wild-type mice (4 versus 88%,P< 0.001). Despite this marked difference in clinical outcome, no differences in numbers of apoptotic cells or presence of histopathologic lesions in the brains of moribund wild-type mice and those of clinically healthy IL-1β−/−mice could be detected. These results suggest that IL-1β deficiency is protective against fatal Sindbis virus infection by a mechanism that does not involve resistance to CNS virus-induced apoptosis or histopathology.
Opposing Effects of Human Immunodeficiency Virus Type 1 Matrix Mutations Support a Myristyl Switch Model of Gag Membrane Targeting
Authors: Paillart Jean-Christophe , Göttlinger Heinrich G.
Published: December 2019

Targeting of the human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55

gag

to the plasma membrane, the site of virus assembly, is primarily mediated by the N-terminal matrix (MA) domain. N-myristylation of MA is essential for the stable association of Pr55

gag

with membranes and for virus assembly. We now show that single amino acid substitutions near the N terminus of MA can dramatically impair assembly without compromising myristylation. Subcellular fractionation demonstrated that Gag membrane binding was compromised to a similar extent as in the absence of the myristyl acceptor site, indicating that the myristyl group was not available for membrane insertion. Remarkably, the effects of the N-terminal modifications could be completely suppressed by second-site mutations in the globular core of MA. The compensatory mutations enhanced Gag membrane binding and increased viral particle yields above wild-type levels, consistent with an increase in the exposure of the myristyl group. Our results support a model in which the compact globular core of MA sequesters the myristyl group to prevent aberrant binding to intracellular membranes, while the N terminus is critical to allow the controlled exposure of the myristyl group for insertion into the plasma membrane.
A Brome Mosaic Virus Intergenic RNA3 Replication Signal Functions with Viral Replication Protein 1a To Dramatically Stabilize RNA In Vivo
Authors: Sullivan Michael L. , Ahlquist Paul
Published: December 2019

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain.Saccharomyces cerevisiaeexpressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast. As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, we have identified thecis-acting sequences required for this effect. We find that 1a-induced stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication. Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous β-globin RNA. Within this intergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and positive-strand RNA3 replication products in yeast expressing 1a and 2a. In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV RNA replication.
The Transmembrane Domain of Hepatitis C Virus Glycoprotein E1 Is a Signal for Static Retention in the Endoplasmic Reticulum
Authors: Cocquerel Laurence , Duvet Sandrine , Meunier Jean-Christophe , Pillez André , Cacan René , Wychowski Czeslaw , Dubuisson Jean
Published: December 2019

Hepatitis C virus (HCV) glycoproteins E1 and E2 assemble to form a noncovalent heterodimer which, in the cell, accumulates in the endoplasmic reticulum (ER). Contrary to what is observed for proteins with a KDEL or a KKXX ER-targeting signal, the ER localization of the HCV glycoprotein complex is due to a static retention in this compartment rather than to its retrieval from the cis-Golgi region. A static retention in the ER is also observed when E2 is expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain (TMD) of E2. Although they do not exclude the presence of an intracellular localization signal in E1, these data do suggest that the TMD of E2 is an ER retention signal for HCV glycoprotein complex. In this study chimeric proteins containing the ectodomain of CD4 or CD8 fused to the C-terminal hydrophobic sequence of E1 were shown to be localized in the ER, indicating that the TMD of E1 is also a signal for ER localization. In addition, these chimeric proteins were not processed by Golgi enzymes, indicating that the TMD of E1 is responsible for true retention in the ER, without recycling through the Golgi apparatus. Together, these data suggest that at least two signals (TMDs of E1 and E2) are involved in ER retention of the HCV glycoprotein complex.
Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
Authors: Urabe Masashi , Hasumi Yoko , Kume Akihiro , Surosky Richard T. , Kurtzman Gary J. , Tobita Kiyotake , Ozawa Keiya
Published: December 2019

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the
trs
(terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a
trs
homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the
trs
homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay,
trs
endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or
trs
nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the
trs
homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.
Identification of CXCR4 Domains That Support Coreceptor and Chemokine Receptor Functions
Authors: Doranz Benjamin J. , Orsini Michael J. , Turner Julie D. , Hoffman Trevor L. , Berson Joanne F. , Hoxie James A. , Peiper Stephen C. , Brass Lawrence F. , Doms Robert W.
Published: December 2019

The interaction of the chemokine stromal cell-derived factor 1 (SDF-1) with its receptor CXCR4 is vital for cell trafficking during development, is capable of inhibiting human immunodeficiency virus type 1 (HIV-1) utilization of CXCR4 as a coreceptor, and has been implicated in delaying disease progression to AIDS in vivo. Because of the importance of this chemokine-chemokine receptor pair to both development and disease, we investigated the molecular basis of the interaction between CXCR4 and its ligands SDF-1 and HIV-1 envelope. Using CXCR4 chimeras and mutants, we determined that SDF-1 requires the CXCR4 amino terminus for binding and activates downstream signaling pathways by interacting with the second extracellular loop of CXCR4. SDF-1-mediated activation of CXCR4 required the Asp-Arg-Tyr motif in the second intracellular loop of CXCR4, was pertussis toxin sensitive, and did not require the distal C-terminal tail of CXCR4. Several CXCR4 mutants that were not capable of binding SDF-1 or signaling still supported HIV-1 infection, indicating that the ability of CXCR4 to function as a coreceptor is independent of its ability to signal. Direct binding studies using the X4 gp120s HXB, BH8, and MN demonstrated the ability of HIV-1 gp120 to bind directly and specifically to the chemokine receptor CXCR4 in a CD4-dependent manner, using a conformationally complex structure on CXCR4. Several CXCR4 variants that did not support binding of soluble gp120 could still function as viral coreceptors, indicating that detectable binding of monomeric gp120 is not always predictive of coreceptor function.
Detection and Induction of Equine Infectious Anemia Virus-Specific Cytotoxic T-Lymphocyte Responses by Use of Recombinant Retroviral Vectors
Authors: Lonning S. M. , Zhang W. , Leib S. R. , McGuire T. C.
Published: December 2019

Cytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and using the gibbon ape leukemia virus (GALV) Env for internalization were efficient at transducing equine kidney (EK) target cells and were effective targets for EIAV-specific CTL lysis. CTL from EIAV-infected horses caused lysis of retroviral vector-transduced EK cells expressing either Gag/Pr or SU in an ELA-A-restricted manner. In contrast, lysis of recombinant vaccinia virus-infected EK cells expressing Gag/Pr and SU/TM was often non-LA-A restricted. Five horses were immunized by direct intramuscular injection with a mixture of retroviral vectors expressing Gag/Pr or SU, and one responded with EIAV-specific CTL. This result indicates that retroviral vector stimulation of CTL in horses needs to be optimized, perhaps by inclusion of appropriate cytokine genes in the constructs. However, the studies demonstrated that retroviral vector-transduced target cells were very effective for in vitro dissection of EIAV-specific CTL responses.
Hepatitis C Virus Core Protein Interacts with Cellular Putative RNA Helicase
Authors: You Li-Ru , Chen Chun-Ming , Yeh Tien-Shun , Tsai Tzung-Yuan , Mai Ru-Tsun , Lin Chi-Hung , Lee Yan-Hwa Wu
Published: December 2019

The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to
trans
-act on several viral or cellular promoters. To get insight into the
trans
-action mechanism of HCV core protein, a yeast two-hybrid cloning system was used for identification of core protein-interacting cellular protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV core protein with this targeted protein. Additionally, the protein’s interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV core protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV core protein. Moreover, transient expression of HCV core protein in human hepatoma HuH-7 cells significantly potentiated the
trans
-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV core protein promotes the
trans
-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf. These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid core protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism. This feature of core protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.
Primary and Secondary Structural Elements Required for Synthesis of Barley Yellow Dwarf Virus Subgenomic RNA1
Authors: Koev Gennadiy , Mohan B. R. , Miller W. Allen
Published: December 2019

Barley yellow dwarf luteovirus (BYDV) generates three 3′-coterminal subgenomic RNAs (sgRNAs) in infected cells. The promoter of sgRNA1 is a putative hot spot for RNA recombination in luteovirus evolution. The sgRNA1 transcription start site was mapped previously to either nucleotide 2670 or nucleotide 2769 of BYDV genomic RNA (gRNA) in two independent studies. Our data support the former initiation site. The boundaries of the sgRNA1 promoter map between nucleotides 2595 and 2692 on genomic RNA. Computer prediction, phylogenetic comparison, and structural probing revealed two stem-loops (SL1 and SL2) in the sgRNA1 promoter region on the negative strand. Promoter function was analyzed by inoculating protoplasts with a full-length infectious clone of the BYDV genome containing mutations in the sgRNA promoter. Because the promoter is located in an essential coding region of the replicase gene, we duplicated it in a nonessential part of the genome from which a new sgRNA was expressed. Mutational analysis revealed that secondary structure, but not the nucleotide sequence, was important at the base of SL1. Regions with both RNA primary and secondary structural features that contributed to transcription initiation were found at the top of SL1. Primary sequence, but not the secondary structure, was required in SL2, which includes the initiation site. Disruption of base pairing near the sgRNA1 start site increased the level of transcription three- to fourfold. We propose that both primary and secondary structures of the sgRNA1 promoter of BYDV play unique roles in sgRNA1 promoter recognition and transcription initiation.
A Proline-Rich Motif within the Matrix Protein of Vesicular Stomatitis Virus and Rabies Virus Interacts with WW Domains of Cellular Proteins: Implications for Viral Budding
Authors: Harty Ronald N. , Paragas Jason , Sudol Marius , Palese Peter
Published: December 2019

The matrix (M) protein of rhabdoviruses has been shown to play a key role in virus assembly and budding; however, the precise mechanism by which M mediates these processes remains unclear. We have associated a highly conserved, proline-rich motif (PPxY or PY motif, where P denotes proline, Y represents tyrosine, and x denotes any amino acid) of rhabdoviral M proteins with a possible role in budding mediated by the M protein. Point mutations that disrupt the PY motif of the M protein of vesicular stomatitis virus (VSV) have no obvious effect on membrane localization of M but instead lead to a decrease in the amount of M protein released from cells in a functional budding assay. Interestingly, the PPxY sequence within rhabdoviral M proteins is identical to that of the ligand which interacts with WW domains of cellular proteins. Indeed, results from two in vitro binding assays demonstrate that amino acids 17 through 33 and 29 through 44, which contain the PY motifs of VSV and rabies virus M proteins, respectively, mediate interactions with WW domains of specific cellular proteins. Point mutations that disrupt the consensus PY motif of VSV or rabies virus M protein result in a significant decrease in their ability to interact with the WW domains. These properties of the PY motif of rhabdovirus M proteins are strikingly analogous to those of the late (L) budding domain identified in the
gag
-specific protein p2b of Rous sarcoma virus. Thus, it is possible that rhabdoviruses may usurp host proteins to facilitate the budding process and that late stages in the budding process of rhabdoviruses and retroviruses may have features in common.
Comparative Characterization of Rep Proteins from the Helper-Dependent Adeno-Associated Virus Type 2 and the Autonomous Goose Parvovirus
Authors: Smith Deborah H. , Ward Peter , Linden R. Michael
Published: December 2019

Adeno-associated viruses (AAVs) are nonautonomous human parvoviruses in that they are dependent on helper functions supplied by other viruses or on genotoxic stimuli for conditions permissive for replication. In the absence of helper, AAV type 2 enters latency by integration into a specific site on human chromosome 19. This feature of AAV, in combination with a lack of pathogenicity, makes AAV an attractive candidate vector for human gene therapy. Goose parvovirus (GPV) is both autonomous and pathogenic yet is highly homologous to AAV. To address the molecular bases for the different viral lifestyles, we compare the AAV and GPV nonstructural proteins, Rep78 and Rep1, respectively. We find that Rep78 and Rep1 possess several biochemical activities in common, including (i) high-affinity DNA binding for sequences that constitute the minimal DNA replication origin; (ii) nucleoside triphosphate-dependent DNA helicase activity; and (iii) origin-specific replication of double-stranded linear DNA. These experiments also establish a specific 38-bp DNA sequence as the minimal GPV DNA replication origin. It is noteworthy that although the proposed Rep binding sites of GPV and AAV are highly similar, Rep1 and Rep78 show a high degree of specificity for their respective origins, in both binding and replication assays. One significant difference was observed; with the minimal replication origin in adenovirus-uninfected extracts, Rep78-mediated replication exhibited low processivity, as previously reported. In contrast, Rep1 efficiently replicated full-length template. Overall, our studies indicate that GPV Rep1 and AAV Rep78 support a comparable mode of replication. Thus, a comparison of the two proteins provides a model system with which to determine the contribution of Rep in the regulation of dependence and autonomy at the level of DNA replication.
Glycoprotein gL-Independent Infectivity of Pseudorabies Virus Is Mediated by a gD-gH Fusion Protein
Authors: Klupp Barbara G. , Mettenleiter Thomas C.
Published: December 2019

Envelope glycoproteins gH and gL, which form a complex, are conserved throughout the family
Herpesviridae
. The gH-gL complex is essential for the fusion between the virion envelope and the cellular cytoplasmic membrane during penetration and is also required for direct viral cell-to-cell spread from infected to adjacent noninfected cells. It has been proposed for several herpesviruses that gL is required for proper folding, intracellular transport, and virion localization of gH. In pseudorabies virus (PrV), glycoprotein gL is necessary for infectivity but is dispensable for virion localization of gH. A virus mutant lacking gL, PrV-ΔgLβ, is defective in entry into target cells, and direct cell-to-cell spread is drastically reduced, resulting in only single or small foci of infected cells (B. G. Klupp, W. Fuchs, E. Weiland, and T. C. Mettenleiter, J. Virol. 71:7687–7695, 1997). We used this limited cell-to-cell spreading ability of PrV-ΔgLβ for serial passaging of cells infected with transcomplemented virus by coseeding with noninfected cells. After repeated passaging, plaque formation was restored and infectivity in the supernatant was observed. One single-plaque isolate, designated PrV-ΔgLPass, was further characterized. To identify the mutation leading to this gL-independent infectious phenotype, Southern and Western blot analyses, radioimmunoprecipitations, and DNA sequencing were performed. The results showed that rearrangement of a genomic region comprising part of the gH gene into a duplicated copy of part of the unique short region resulted in a fusion fragment predicted to encode a protein consisting of the N-terminal 271 amino acids of gD fused to the C-terminal 590 residues of gH. Western blotting and radioimmunoprecipitation with gD- and gH-specific antibodies verified the presence of a gDH fusion protein. To prove that this fusion protein mediates infectivity of PrV-ΔgLPass, cotransfection of PrV-ΔgLβ DNA with the cloned fusion fragment was performed, and a cell line, Nde-67, carrying the fusion gene was established. After cotransfection, infectious gL-negative PrV was recovered, and propagation of PrV-ΔgLβ on Nde-67 cells produced infectious virions. Thus, a gDH fusion polypeptide can compensate for function of the essential gL in entry and cell-to-cell spread of PrV.
Substitutions in the Receptor-Binding Domain of the Avian Sarcoma and Leukosis Virus Envelope Uncouple Receptor-Triggered Structural Rearrangements in the Surface and Transmembrane Subunits
Authors: Damico Rachel , Rong Lijun , Bates Paul
Published: December 2019

The retrovirus avian sarcoma and leukosis virus (ASLV) enters cells via pH-independent membrane fusion. This reaction is catalyzed by the viral glycoprotein Env, composed of a membrane-distal subunit, SU, and a membrane-anchored subunit, TM. Previous mutational analysis of a variable region, central within the SU subunit, indicates that this region constitutes part of the receptor-binding domain for subgroup A envelope (EnvA) and furthermore that basic residues (R210, R213, R223, R224, and K227) within this region are critical determinants of efficient ASLV infection. Substitutions of these basic residues exert effects on both receptor binding and postbinding events in EnvA-mediated entry. In this study, we performed biochemical analysis of the EnvA protein from three of the receptor-binding domain mutants (R213A/K227A, R213A/R223A/R224A, and R213S) to define the role of this domain in early molecular events in the entry pathway. Protease sensitivity assays demonstrated that receptor binding was sufficient to trigger conformational changes in the SU subunit of mutants R213A/K227A and R213S similar to those in the wild-type EnvA, while R213A/R223A/R224A was constitutively sensitive to protease. In contrast, all three receptor-binding domain mutants disrupted receptor-triggered conversion of EnvA to an active, membrane-binding conformation as assessed by liposome flotation assays. Our results demonstrate that mutations in the receptor-binding site can dissociate receptor-triggered conformational changes in the SU subunit from membrane binding. Furthermore, they suggest that communication between the receptor-binding subunit, SU, and the fusogenic subunit, TM, is crucial for efficient activation of the fusogenic state of EnvA. Analysis of these mutants continues earlier observations that binding to the cellular receptor provides the trigger for efficient activation of this pH-independent viral envelope protein.
Yellow Fever/Japanese Encephalitis Chimeric Viruses: Construction and Biological Properties
Authors: Chambers Thomas J. , Nestorowicz Ann , Mason Peter W. , Rice Charles M.
Published: December 2019

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA
14
-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA
14
-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10
6
PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.
The Simian Virus 40 Small-t and Large-T Antigens Jointly Regulate Cell Cycle Reentry in Human Fibroblasts
Authors: Porrás Analía , Gaillard Stéphanie , Rundell Kathleen
Published: December 2019

Focus formation in human diploid fibroblasts (HDF cells) is known to require both the simian virus 40 (SV40) large-T and small-t antigens. Similarly, both SV40 proteins were required to stimulate confluent, density-arrested HDF cells to reenter the cell cycle. This study used defective recombinant adenoviruses to examine the roles of the individual SV40 proteins in altering specific steps in the cell cycle. Small-t antigen and, to a lesser extent, large-T antigen increased the level of the S phase cyclin cyclin A but without increasing the activity of associated cyclin kinases unless the two SV40 proteins were coexpressed. The absence of kinase activity reflected the presence in density-arrested cells of high levels of the cyclin-dependent kinase inhibitors p21
WAF1
and p27
KIP1
. We report here that expression of SV40 large-T antigen reduced levels of p21
WAF1
, while expression of small-t antigen was required to decrease p27
KIP1
. The separate effects of large-T and small-t antigens on these two inhibitors may explain the joint requirement for the two proteins to drive cell cycle reentry of HDF cells and ultimately transform these cells.
The Serine Protease and RNA-Stimulated Nucleoside Triphosphatase and RNA Helicase Functional Domains of Dengue Virus Type 2 NS3 Converge within a Region of 20 Amino Acids
Authors: Li Haitao , Clum Stephen , You Shihyun , Ebner Kurt E. , Padmanabhan R.
Published: December 2019

NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in
Escherichia coli
and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues,
184
RKRK→QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues
184
RKRK, plays an important role for the RNA-stimulated NTPase activity.