bluuebunny/crossref_metadata_2025 huggingface dataset:
Reovirus Virion-Like Particles Obtained by Recoating Infectious Subvirion Particles with Baculovirus-Expressed ς3 Protein: an Approach for Analyzing ς3 Functions during Virus Entry
Authors: Jané-Valbuena Judit , Nibert Max L. , Spencer Stephan M. , Walker Stephen B. , Baker Timothy S. , Chen Ya , Centonze Victoria E. , Schiff Leslie A.
Published: December 2019

Structure-function studies with mammalian reoviruses have been limited by the lack of a reverse-genetic system for engineering mutations into the viral genome. To circumvent this limitation in a partial way for the major outer-capsid protein ς3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed ς3 protein to infectious subvirion particles (ISVPs) that lack ς3. A level of ς3 binding approaching 100% of that in native virions was routinely achieved. The ς3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image reconstruction. rcISVPs retained full infectivity in murine L cells, allowing their use to study ς3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to exponential growth and in being inhibited from entering cells by either the weak base NH
4
Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimicked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the viral mRNAs. Last, rcISVPs behaved like virions in showing minor loss of infectivity at 52°C. Since rcISVPs contain virion-like levels of ς3 but contain outer-capsid protein μ1/μ1C mostly cleaved at the δ-φ junction as in ISVPs, the fact that rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that ς3, and not the δ-φ cleavage of μ1/μ1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of protein functions in reovirus entry (an approach that we call recoating genetics), we used chimeric ς3 proteins to localize the primary determinants of a strain-dependent difference in ς3 cleavage rate to a carboxy-terminal region of the ISVP-bound protein.
Early Region 1 Transforming Functions Are Dispensable for Mammary Tumorigenesis by Human Adenovirus Type 9
Authors: Thomas Darby L. , Shin Sook , Jiang Bernard H. , Vogel Hannes , Ross Margery A. , Kaplitt Michael , Shenk Thomas E. , Javier Ronald T.
Published: December 2019

Some human adenoviruses are tumorigenic in rodents. Subgroup A and B human adenoviruses generally induce sarcomas in both male and female animals, and the gene products encoded within viral early region 1 (E1 region) are both necessary and sufficient for this tumorigenicity. In contrast, subgroup D human adenovirus type 9 (Ad9) induces estrogen-dependent mammary tumors in female rats and requires the E4 region-encoded ORF1 oncoprotein for its tumorigenicity. Considering the established importance of the viral E1 region for tumorigenesis by adenoviruses, we investigated whether this viral transcription unit is also necessary for Ad9 to generate mammary tumors. The nucleotide sequence of the Ad9 E1 region indicated that the gene organization and predicted E1A and E1B polypeptides of Ad9 are closely related to those of other human adenovirus E1 regions. In addition, an Ad9 E1 region plasmid demonstrated focus-forming activity in both low-passage-number and established rat embryo fibroblasts, whereas a large deletion within either the E1A or E1B gene of this plasmid diminished transforming activity. Surprisingly, we found that introducing the same transformation-inactivating E1A and E1B deletions into Ad9 results in mutant viruses that retain the ability to elicit mammary tumors in rats. These results are novel in showing that Ad9 represents a unique oncogenic adenovirus in which the E4 region, rather than the E1 region, encodes the major oncogenic determinant in the rat.
Requirements for Measles Virus Induction of RANTES Chemokine in Human Astrocytoma-Derived U373 Cells
Authors: Noe Katherine H. , Cenciarelli Cristina , Moyer Sue A. , Rota Paul A. , Shin Moon L.
Published: December 2019

Interferons and chemokines play a critical role in regulating the host response to viral infection. Measles virus, a member of theParamyxoviridaefamily, induces RANTES expression by astrocytes. We have examined the mechanism of this induction in U373 cells derived from a human astrocytoma. RANTES was induced in a dose- and time-dependent manner by measles virus infection. Inhibition of receptor binding by the anti-CD46 antibody TRA-2.10 and of virus-membrane fusion by the tripeptide X-Phe-Phe-Gly reduced RANTES expression. Formalin-inactivated virus, which can bind but not fuse, and extensively UV-irradiated virus, which can bind and fuse, were both ineffective. Therefore, virus binding to the cellular receptor CD46 and subsequent membrane fusion were necessary, but not sufficient, to induce RANTES. UV irradiation of virus for less than 10 min proportionally inhibited viral transcription and RANTES expression. RANTES induction was decreased in infected cells treated with ribavirin, which inhibits measles virus transcription. However, RANTES mRNA was superinduced by measles virus in the presence of cycloheximide. These data suggest that partial transcription of the viral genome is sufficient and necessary for RANTES induction, whereas viral protein synthesis and replication are not required. This hypothesis was supported by the fact that RANTES was induced through transient expression of the measles virus nucleocapsid gene but not by measles genes encoding P or L proteins or by leader RNA in A549 cells. Thus, transcription of specific portions of measles virus RNA, such as the nucleocapsid gene, appears able to generate the specific signaling required to induce RANTES gene expression.
Distinct Attenuation Phenotypes Caused by Mutations in the Translational Starting Window of Theiler’s Murine Encephalomyelitis Virus
Authors: Pilipenko Evgeny V. , Viktorova Ekaterina G. , Khitrina Elena V. , Maslova Svetlana V. , Jarousse Nadine , Brahic Michel , Agol Vadim I.
Published: December 2019

Upon initiation of translation of picornavirus RNA, the ribosome is believed to bind the internal ribosome entry site of the template and then to form a productive complex with a downstream RNA segment, the starting window. The presence or absence of an AUG triplet within the starting window of the RNA of Theiler’s murine encephalomyelitis virus (a picornavirus) is known to modulate its neurovirulence. In this study, mutants of this virus in which the starting windows, lying upstream of the viral polyprotein reading frame, had AUGs with different nonoptimal contexts were engineered. Upon intracerebral inoculation of mice, the mutants proved to be partially attenuated, as judged by a significant increase in the dose causing paralysis in 50% of the animals (PD
50
). Mutants with similar PD
50
s might differ from one another by eliciting either a severe, fatal tetraplegy or only mild, recoverable neurologic lesions. Some of the mutants triggered a chronic inflammatory reaction in the white matter of the spinal cord in the absence of detectable viral RNA or antigen. Thus, point mutations changing the context of an AUG within the starting window outside the polyprotein reading frame may differently affect the morbidity and mortality caused by a viral infection and may result in distinct attenuation phenotypes.
Visualization of Tegument-Capsid Interactions and DNA in Intact Herpes Simplex Virus Type 1 Virions
Authors: Zhou Z. Hong , Chen Dong Hua , Jakana Joanita , Rixon Frazer J. , Chiu Wah
Published: December 2019

Herpes simplex virus type 1 virions were examined by electron cryomicroscopy, allowing the three-dimensional structure of the infectious particle to be visualized for the first time. The capsid shell is identical to that of B-capsids purified from the host cell nucleus, with the exception of the penton channel, which is closed. The double-stranded DNA genome is organized as regularly spaced (∼26 Å) concentric layers inside the capsid. This pattern suggests a spool model for DNA packaging, similar to that for some bacteriophages. The bulk of the tegument is not icosahedrally ordered. However, a small portion appears as filamentous structures around the pentons, interacting extensively with the capsid. Their locations and interactions suggest possible roles for the tegument proteins in regulating DNA transport through the penton channel and binding to cellular transport proteins during viral infection.
ERV-L Elements: a Family of Endogenous Retrovirus-Like Elements Active throughout the Evolution of Mammals
Authors: Bénit Laurence , Lallemand Jean-Baptiste , Casella Jean-François , Philippe Hervé , Heidmann Thierry
Published: December 2019

We have previously identified in the human genome a family of 200 endogenous retrovirus-like elements, the HERV-L elements, disclosing similarities with the foamy retroviruses and which might be the evolutionary intermediate between classical intracellular retrotransposons and infectious retroviruses. Southern blot analysis of a large series of mammalian genomic DNAs shows that HERV-L-related elements—so-called ERV-L—are present among all placental mammals, suggesting that ERV-L elements were already present at least 70 million years ago. Most species exhibit a low copy number of ERV-L elements (from 10 to 30), while simians (not prosimians) and mice (not rats) have been subjected to bursts resulting in increases in the number of copies up to 200. The burst of copy number in primates can be dated to shortly after the prosimian and simian branchpoint, 45 to 65 million years ago, whereas murine species have been subjected to two much more recent bursts (less than 10 million years ago), occurring after the
Mus/Rattus
split. We have amplified and sequenced 360-bp ERV-L internal fragments of the highly conserved
pol
gene from a series of 22 mammalian species. These sequences exhibit high percentages of identity (57 to 99%) with the murine fully coding MuERV-L element. Phylogenetic analyses allowed the establishment of a plausible evolutionary scheme for ERV-L elements, which accounts for the high level of sequence conservation and the widespread dispersion among mammals.
Acute and Persistent Infection of Human Neural Cell Lines by Human Coronavirus OC43
Authors: Arbour Nathalie , Côté Geneviève , Lachance Claude , Tardieu Marc , Cashman Neil R. , Talbot Pierre J.
Published: December 2019

Human coronaviruses (HuCV) are recognized respiratory pathogens. Data accumulated by different laboratories suggest their neurotropic potential. For example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the OC43 strain of HuCV (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800–806, 1997). We speculate that the neurotropism of HuCV will lead to persistence within the central nervous system, as was observed for murine coronaviruses. As a first step in the verification of our hypothesis, we have characterized the susceptibility of various human neural cell lines to infection by HuCV-OC43. Viral antigen, infectious virus progeny, and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, oligodendrocytic MO3.13, and the CHME-5 immortalized fetal microglial cell lines, were all susceptible to an acute infection by HuCV-OC43. Viral antigen and RNA and release of infectious virions were observed during persistent HuCV-OC43 infections (∼130 days of culture) of U-87 MG, U-373 MG, MO3.13, and H4 cell lines. Nucleotide sequences of RNA encoding the putatively hypervariable viral S1 gene fragment obtained after 130 days of culture were compared to that of initial virus input. Point mutations leading to amino acid changes were observed in all persistently infected cell lines. Moreover, an in-frame deletion was also observed in persistently infected H4 cells. Some point mutations were observed in some molecular clones but not all, suggesting evolution of the viral population and the emergence of viral quasispecies during persistent infection of H4, U-87 MG, and MO3.13 cell lines. These results are consistent with the potential persistence of HuCV-OC43 in cells of the human nervous system, accompanied by the production of infectious virions and molecular variation of viral genomic RNA.
A Herpesvirus Ribosome-Associated, RNA-Binding Protein Confers a Growth Advantage upon Mutants Deficient in a GADD34-Related Function
Authors: Mulvey Matthew , Poppers Jeremy , Ladd Alison , Mohr Ian
Published: December 2019

The herpes simplex virus type 1 γ34.5 gene product and the cellular GADD34 protein both contain similar domains that can regulate the activity of eukaryotic initiation factor 2 (eIF2), a critical translation initiation factor. Viral mutants that lack the GADD34-related function grow poorly on a variety of malignant human cells, as activation of the cellular PKR kinase leads to the accumulation of inactive, phosphorylated eIF2 at late times postinfection. Termination of translation prior to the completion of the viral reproductive cycle leads to impaired growth. Extragenic suppressors that regain the ability to synthesize proteins efficiently in the absence of the viral GADD34-related function have been isolated. These suppressor alleles are dominant in
trans
and affect the steady-state accumulation of several viral mRNA species. We demonstrate that deregulated expression of Us11, a virus-encoded RNA-binding, ribosome-associated protein is necessary and sufficient to confer a growth advantage upon viral mutants that lack a GADD34-related function. Ectopic expression of Us11 reduces the accumulation of the activated cellular PKR kinase and allows for sustained protein synthesis. Thus, an RNA-binding, ribosome-associated protein (Us11) and a GADD34-related protein (γ34.5) both function in a signal pathway that regulates translation by modulating eIF2 phosphorylation.
The Equine Herpesvirus 1 U
S
2 Homolog Encodes a Nonessential Membrane-Associated Virion Component
Authors: Meindl Alexandra , Osterrieder Nikolaus
Published: December 2019

Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 U
S
2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 U
S
2 protein specifically detected a protein with an
M
r
of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-
M
r
U
s
2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-
M
r
U
s
2 polypeptide. Irrespective of its size, the U
S
2 protein was incorporated into virions. The EHV-1 U
S
2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 U
S
2 protein or to a truncated U
S
2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 U
S
2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the U
S
2 protein in the viral envelope and plasma membrane of infected cells, a U
S
2-negative RacL11 mutant (L11ΔU
S
2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a U
S
2-repaired virus. After infection of BALB/c mice with L11ΔU
S
2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 U
S
2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.
Will Multiple Coreceptors Need To Be Targeted by Inhibitors of Human Immunodeficiency Virus Type 1 Entry?
Authors: Zhang Yi-jun , Moore John P.
Published: December 2019

Despite being able to use the Bonzo coreceptor as efficiently as CCR5 in transfected cells, pediatric human immunodeficiency virus type 1 isolate P6 was unable to replicate in peripheral blood mononuclear cells (PBMC) lacking the CCR5 receptor. Furthermore, its replication in wild-type PBMC was completely inhibited by inhibitors of CCR5-mediated entry. Similarly, maternal isolate M6 could use CCR5, CXCR4, Bonzo, and other coreceptors in transfected cells but was completely sensitive to inhibitors of CCR5- and CXCR4-mediated entry when grown in PBMC. The ability of these viruses to use coreceptors in addition to CCR5 and CXCR4 in vitro was, therefore, irrelevant to their drug sensitivity in primary cells. We argue that CCR5 and CXCR4 should remain the primary targets for antiviral drug development, pending strong evidence to the contrary.
Direct Evidence that the Poly(A) Tail of Influenza A Virus mRNA Is Synthesized by Reiterative Copying of a U Track in the Virion RNA Template
Authors: Poon Leo L. M. , Pritlove David C. , Fodor Ervin , Brownlee George G.
Published: December 2019

The poly(A) tail of influenza virus mRNA is thought to be synthesized by reiterative copying of the U track near the 5′ end of the virion RNA template. This has been widely accepted as a plausible hypothesis, but until now there has been no direct experimental evidence for it. Here, we report such direct evidence based on the fact that (i) replacing the U track with an A track directs synthesis of products with poly(U) tails, both in vitro and in vivo, and (ii) interrupting the U track abolishes polyadenylation in vitro.
Infectious Pancreatic Necrosis Virus: Identification of a VP3-Containing Ribonucleoprotein Core Structure and Evidence for O-Linked Glycosylation of the Capsid Protein VP2
Authors: Hjalmarsson Anna , Carlemalm Eric , Everitt Einar
Published: December 2019

Virions of infectious pancreatic necrosis virus (IPNV) were completely disintegrated upon dialysis against salt-free buffers. Direct visualization of such preparations by electron microscopy revealed 5.0- to 6.5-nm-thick entangled filaments. By using a specific colloidal gold immunolabeling technique, these structures were shown to contain the viral protein VP3. Isolation by sucrose gradient centrifugation of the filaments, followed by serological analysis, demonstrated that the entire VP3 content of the virion was recovered together with the radiolabeled genomic material forming the unique threadlike ribonucleoprotein complexes. In a sensitive blotting assay, the outer capsid component of IPNV, i.e., the major structural protein VP2, was shown to specifically bind lectins recognizing sugar moieties of
N
-acetylgalactosamine, mannose, and fucose. Three established metabolic inhibitors of N-linked glycosylation did not prevent addition of sugar residues to virions, and enzymatic deglycosylation of isolated virions using
N
-glycosidase failed to remove sugar residues of VP2 recognized by lectins. However, gentle alkaline β elimination clearly reduced the ability of lectins to recognize VP2. These results suggest that the glycosylation of VP2 is of the O-linked type when IPNV is propagated in RTG-2 cells.
Highly Potent RANTES Analogues either Prevent CCR5-Using Human Immunodeficiency Virus Type 1 Infection In Vivo or Rapidly Select for CXCR4-Using Variants
Authors: Mosier Donald E. , Picchio Gastón R. , Gulizia Richard J. , Sabbe Rebecca , Poignard Pascal , Picard Laurent , Offord Robin E. , Thompson Darren A. , Wilken Jill
Published: December 2019

The natural ligands for the CCR5 chemokine receptor, macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and RANTES (regulated on T-cell activation, normal T-cell expressed and secreted), are known to inhibit human immunodeficiency virus (HIV) entry, and N-terminally modified RANTES analogues are more potent than native RANTES in blocking infection. However, potent CCR5 blocking agents may select for HIV-1 variants that use alternative coreceptors at less than fully inhibitory concentrations. In this study, two N-terminal chemical modifications of RANTES produced by total synthesis, aminooxypentane (AOP)-RANTES[2-68] and
N
-nonanoyl (NNY)-RANTES[2-68], were tested for their ability to prevent HIV-1 infection and to select for coreceptor switch variants in the human peripheral blood lymphocyte-SCID mouse model. Mice were infected with a CCR5-using HIV-1 isolate that requires only one or two amino acid substitutions to use CXCR4 as a coreceptor. Even though it achieved lower circulating concentrations than AOP-RANTES (75 to 96 pM as opposed to 460 pM under our experimental conditions), NNY-RANTES was more effective in preventing HIV-1 infection. However, in a subset of treated mice, these levels of NNY-RANTES rapidly selected viruses with mutations in the V3 loop of envelope that altered coreceptor usage. These results reinforce the case for using agents that block all significant HIV-1 coreceptors for effective therapy.
Characterization of an E1A-CBP Interaction Defines a Novel Transcriptional Adapter Motif (TRAM) in CBP/p300
Authors: O’Connor Mark J. , Zimmermann Holger , Nielsen Søren , Bernard Hans-Ulrich , Kouzarides Tony
Published: December 2019

The adenovirus E1A protein subverts cellular processes to induce mitotic activity in quiescent cells. Important targets of E1A include members of the transcriptional adapter family containing CBP/p300. Competition for CBP/p300 binding by various cellular transcription factors has been suggested as a means of integrating different signalling pathways and may also represent a potential mechanism by which E1A manipulates cell fate. Here we describe the characterization of the interaction between E1A and the C/H3 region of CBP. We define a novel conserved 12-residue transcriptional adapter motif (TRAM) within CBP/p300 that represents the binding site for both E1A and numerous cellular transcription factors. We also identify a sequence (FPESLIL) within adenovirus E1A that is required to bind the CBP TRAM. Furthermore, an E1A peptide containing the FPESLIL sequence is capable of preventing the interaction between CBP and TRAM-binding transcription factors, such as p53, E2F, and TFIIB, thus providing a molecular model for E1A action. As an in vivo demonstration of this model, we used a small region of CBP containing a functional TRAM that can bind to the p53 protein. The CBP TRAM binds p53 sequences targeted by the cellular regulator MDM2, and we demonstrate that an MDM2-p53 interaction can be disrupted by the CBP TRAM, leading to stabilization of cellular p53 levels and the activation of p53-dependent transcription. Transcriptional activation of p53 by the CBP TRAM is abolished by wild-type E1A but not by a CBP-binding-deficient E1A mutant.
Identification of a Novel GC-Rich 113-Nucleotide Region To Complete the Circular, Single-Stranded DNA Genome of TT Virus, the First Human Circovirus
Authors: Miyata Hironori , Tsunoda Hiroaki , Kazi Aslamuzzaman , Yamada Akio , Khan Mohammed Ali , Murakami Jun , Kamahora Toshio , Shiraki Kazuo , Hino Shigeo
Published: December 2019

The sequence data (H. Okamoto et al., Hepatol. Res. 10:1–16, 1998) of a newly discovered single-stranded DNA virus, TT virus (TTV), showed that it did not have the terminal structure typical of a parvovirus. Elucidation of the complete genome structure was necessary to understand the nature of TTV. We obtained a 1.0-kb amplified product from serum samples of four TTV carriers by an inverted, nested long PCR targeted for nucleotides (nt) 3025 to 3739 and 1 to 216 of TTV. The sequence of a clone obtained from serum sample TA278 was compared with those registered in GenBank. The complete circular TTV genome contained a novel sequence of 113 nt (nt 3740 to 3852 [=0]) in between the known 3′- and 5′-end arms, forming a 117-nt GC-rich stretch (GC content, 90.6% at nt 3736 to 3852). We found a 36-nt stretch (nt 3816 to 3851) with an 80.6% similarity to chicken anemia virus (CAV) (nt 2237 to 2272 of M55918), a vertebrate circovirus. A putative SP-1 site was located at nt 3834 to 3839, followed by a TATA box at nt 85 to 90, the first initiation codon of a putative VP2 at nt 107 to 109, the termination codon of a putative VP1 at nt 2899 to 2901, and a poly(A) signal at nt 3073 to 3078. The arrangement was similar to that of CAV. Furthermore, several AP-2 and ATF/CREB binding sites and an NF-κB site were arranged around the GC-rich region in both TTV and CAV. The data suggested that TTV is circular and similar to CAV in its genomic organization, implying that TTV is the first human circovirus.
Retroviral Insertions inEvi12, a Novel Common Virus Integration Site Upstream ofTra1/Grp94, Frequently Coincide with Insertions in the Gene Encoding the Peripheral Cannabinoid ReceptorCnr2
Authors: Valk Peter J. M. , Vankan Yolanda , Joosten Marieke , Jenkins Nancy A. , Copeland Neal G. , Löwenberg Bob , Delwel Ruud
Published: December 2019

The common virus integration site (VIS)Evi11was recently identified within the gene encoding the hematopoietic G-protein-coupled peripheral cannabinoid receptorCnr2(also referred to asCb2). Here we show thatCnr2is a frequent target (12%) for insertion of Cas-Br-M murine leukemia virus (MuLV) in primary tumors in NIH/Swiss mice. Multiple provirus insertions inEvi11were cloned and shown to be located within the 3′ untranslated region of the candidate proto-oncogeneCnr2. These results suggest that proviral insertion in theCnr2gene is an important step in Cas-Br-M MuLV-induced leukemogenesis in NIH/Swiss mice. To isolateEvi11/Cnr2collaborating proto-oncogenes, we searched for novel common VISs in the Cas-Br-M MuLV-induced primary tumors and identified a novel frequent common VIS,Evi12(14%). Interestingly, 54% of theEvi11/Cnr2-rearranged primary tumors contained insertions inEvi12as well, which suggests cooperative action of the target genes in these two common VISs in leukemogenesis. By interspecific backcross analysis it was shown thatEvi12resides on mouse chromosome 10 in a region that shares homology with human chromosomes 12q and 19p. Sequence analysis demonstrated thatEvi12is located upstream of the gene encoding the molecular chaperoneTra1/Grp94, which was previously mapped to mouse chromosome 10 and human chromosome 12q22–24. Thus,Tra1/Grp94is a candidate target gene for retroviral activation or inactivation inEvi12. However, Northern and Western blot analyses did not provide evidence that proviral insertion had altered the expression ofTra1/Grp94. Additional studies are required to determine whetherTra1/Grp94or another candidate proto-oncogene inEvi12is involved in leukemogenesis.
Opposite Effects of SDF-1 on Human Immunodeficiency Virus Type 1 Replication
Authors: Maréchal Valérie , Arenzana-Seisdedos Fernando , Heard Jean-Michel , Schwartz Olivier
Published: December 2019

The α-chemokine SDF-1 binds CXCR4, a coreceptor for human immunodeficiency virus type 1 (HIV-1), and inhibits viral entry mediated by this receptor. Since chemokines are potent chemoattractants and activators of leukocytes, we examined whether the stimulation of HIV target cells by SDF-1 affects the replication of virus with different tropisms. We observed that SDF-1 inhibited the entry of X4 strains and increased the infectivity of particles bearing either a CCR5-tropic HIV-1 envelope or a vesicular stomatitis virus G envelope. In contrast to the inhibitory effect of SDF-1 on X4 strains, which is at the level of entry, the stimulatory effect does not involve envelope-receptor interactions or proviral DNA synthesis. Rather, we observed an increased ability of Tat to transactivate the HIV-1 long terminal repeat in the presence of the chemokine. Therefore, the effects of SDF-1 on the HIV-1 life cycle can be multiple and opposite, including both an inhibition of viral entry and a stimulation of proviral gene expression.
Enrichment of a Precore-Minus Mutant of Duck Hepatitis B Virus in Experimental Mixed Infections
Authors: Zhang Yong-Yuan , Summers Jesse
Published: December 2019

A precore-deficient mutant of duck hepatitis B virus (DHBV) produced by site-directed mutagenesis was tested for its ability to compete with wild-type virus in a mixed infection of 3-day-old ducklings. The mutation was shown to produce acis-acting defect, resulting in a replication rate that was about one-half that of wild-type virus. Accordingly, wild-type virus was rapidly selected during the spread of infection. During the chronic phase of the infection, however, two selection patterns were seen. In 4 of 10 ducks, the wild-type virus slowly replaced the precore mutant. In another four ducks, the precore mutant virus slowly replaced the wild-type virus. In the remaining two ducklings, ratios of wild-type and precore mutant virus fluctuated, with wild-type virus slowly predominating. The replacement of wild-type virus was not due to the emergence of a rapidly replicating variant of the precore mutant, since genomes cloned from the infected ducks retained their original replication defect. Replacement of wild-type virus, however, correlated with elevated anti-core antibody titers, which continued to increase with time. The selection of a precore-negative strain of DHBV may be analogous to the selection for precore mutants of HBV during chronic hepatitis in humans.
Shared Usage of the Chemokine Receptor CXCR4 by Primary and Laboratory-Adapted Strains of Feline Immunodeficiency Virus
Authors: Richardson Jennifer , Pancino Gianfranco , Merat Rastine , Leste-Lasserre Thierry , Moraillon Anne , Schneider-Mergener Jens , Alizon Marc , Sonigo Pierre , Heveker Nikolaus
Published: December 2019

Strains of the feline immunodeficiency virus (FIV) presently under investigation exhibit distinct patterns of in vitro tropism. In particular, the adaptation of FIV for propagation in Crandell feline kidney (CrFK) cells results in the selection of strains capable of forming syncytia with cell lines of diverse species origin. The infection of CrFK cells by CrFK-adapted strains appears to require the chemokine receptor CXCR4 and is inhibited by its natural ligand, stromal cell-derived factor 1α (SDF-1α). Here we found that inhibitors of CXCR4-mediated infection by human immunodeficiency virus type I (HIV-1), such as the bicyclam AMD3100 and short peptides derived from the amino-terminal region of SDF-1α, also blocked infection of CrFK by FIV. Nevertheless, we observed differences in the ranking order of the peptides as inhibitors of FIV and HIV-1 and showed that such differences are related to the species origin of CXCR4 and not that of the viral envelope. These results suggest that, although the envelope glycoproteins of FIV and HIV-1 are substantially divergent, FIV and HIV-1 interact with CXCR4 in a highly similar manner. We have also addressed the role of CXCR4 in the life cycle of primary isolates of FIV. Various CXCR4 ligands inhibited infection of feline peripheral blood mononuclear cells (PBMC) by primary FIV isolates in a concentration-dependent manner. These ligands also blocked the viral transduction of feline PBMC by pseudotyped viral particles when infection was mediated by the envelope glycoprotein of a primary FIV isolate but not by the G protein of vesicular stomatitis virus, indicating that they act at an envelope-mediated step and presumably at viral entry. These findings strongly suggest that primary and CrFK-adapted strains of FIV, despite disparate in vitro tropisms, share usage of CXCR4.
Replicative Fitness of Protease Inhibitor-Resistant Mutants of Human Immunodeficiency Virus Type 1
Authors: Martinez-Picado Javier , Savara Anu V. , Sutton Lorraine , D’Aquila Richard T.
Published: December 2019

The relative replicative fitness of human immunodeficiency virus type 1 (HIV-1) mutants selected by different protease inhibitors (PIs) in vivo was determined. Each mutant was compared to wild type (WT), NL4-3, in the absence of drugs by several methods, including clonal genotyping of cultures infected with two competing viral variants, kinetics of viral antigen production, and viral infectivity/virion particle ratios. A nelfinavir-selected protease D30N substitution substantially decreased replicative capacity relative to WT, while a saquinavir-selected L90M substitution moderately decreased fitness. The D30N mutant virus was also outcompeted by the L90M mutant in the absence of drugs. A major natural polymorphism of the HIV-1 protease, L63P, compensated well for the impairment of fitness caused by L90M but only slightly improved the fitness of D30N. Multiply substituted indinavir-selected mutants M46I/L63P/V82T/I84V and L10R/M46I/L63P/V82T/I84V were just as fit as WT. These results indicate that the mutations which are usually initially selected by nelfinavir and saquinavir, D30N and L90M, respectively, impair fitness. However, additional mutations may improve the replicative capacity of these and other drug-resistant mutants. Hypotheses based on the greater fitness impairment of the nelfinavir-selected D30N mutant are suggested to explain observations that prolonged responses to delayed salvage regimens, including alternate PIs, may be relatively common after nelfinavir failure.
Genetic Studies Exposing the Splicing Events Involved in Herpes Simplex Virus Type 1 Latency-Associated Transcript Production during Lytic and Latent Infection
Authors: Alvira Mauricio R. , Goins William F. , Cohen Justus B. , Glorioso Joseph C.
Published: December 2019

Herpes simplex virus type 1 (HSV-1) establishes latency in sensory neurons, a state in which the viral lytic genes are silenced and only the latency locus is transcriptionally active, producing the 2.0- and 1.5-kb latency-associated transcripts (LATs). Previous experimental evidence indicates that the LATs are stable introns, and it has been reported that LAT formation is abolished by debilitating substitution mutations in the predicted splice sites during lytic infection but not latency (J. L. Arthur et al., J. Gen. Virol. 79:107–116, 1998). We have independently studied a set of deletion mutations to explore the roles of the proposed splice sites during lytic and latent infection. HSV-1 mutant viruses missing the invariant intron-terminal 5′-G(T/C) or 3′-AG dinucleotides were analyzed for LAT formation during lytic infection in vitro, when only the 2-kb LAT is produced, and during latency in mouse trigeminal ganglia, where both LATs are expressed. Northern blot analysis of total RNAs from different productively infected cell lines showed that the lytic (2-kb) LAT was not expressed by the various splice site deletion mutants. In vivo studies using a mouse eye model of latency similarly showed that the latent (2- and 1.5-kb) LATs were not expressed by the mutants. PCR analysis with primers flanking the LAT sequence revealed the expected splice junction for LAT excision in RNA from sensory neurons latently infected with wild-type but not mutant virus. Using a virus mutant deleted in the splicing signals flanking the 556-bp region of LAT whose absence distinguishes the 1.5- and 2-kb LATs, we observed selective elimination of 1.5-kb LAT expression in latency, supporting previous suggestions that the internal region is removed by splicing. Taken together, these results demonstrate that the 2-kb LAT is formed during both lytic and latent infection by splicing at the predicted splice sites and that an additional splicing event is involved in the latency-restricted production of the 1.5-kb LAT. We have also mapped the 3′ end of the lytic 2-kb LAT and discuss our results in the context of previous models addressing the unusual stability of the LATs.
Role of Cellular Tumor Necrosis Factor Receptor-Associated Factors in NF-κB Activation and Lymphocyte Transformation by Herpesvirus Saimiri STP
Authors: Lee Heuiran , Choi Joong-Kook , Li Mengtao , Kaye Ken , Kieff Elliott , Jung Jae U.
Published: December 2019

The STP oncoproteins of the herpesvirus saimiri (HVS) subgroup A strain 11 and subgroup C strain 488 are now found to be stably associated with tumor necrosis factor receptor-associated factor (TRAF) 1, 2, or 3. Mutational analyses identified residues of PXQXT/S in STP-A11 as critical for TRAF association. In addition, a somewhat divergent region of STP-C488 is critical for TRAF association. Mutational analysis also revealed that STP-C488 induced NF-κB activation that was correlated with its ability to associate with TRAFs. The HVS STP-C488 P10→R mutant was deficient in human T-lymphocyte transformation to interleukin-2-independent growth but showed wild-type phenotype for marmoset T-lymphocyte transformation in vitro and in vivo. The STP-C488 P10→R mutant was also defective in Rat-1 fibroblast transformation, and fibroblast cell transformation was blocked by a TRAF2 dominant-negative mutant. These data implicate TRAFs in STP-C488-mediated transformation of human lymphocytes and rodent fibroblasts. Other factors are implicated in immortalization of common marmoset T lymphocytes and may also be critical in the transformation of human lymphocytes and rodent fibroblasts.
Alpha Interferon Inhibits Human Herpesvirus 8 (HHV-8) Reactivation in Primary Effusion Lymphoma Cells and Reduces HHV-8 Load in Cultured Peripheral Blood Mononuclear Cells
Authors: Monini Paolo , Carlini Francesca , Stürzl Michael , Rimessi Paola , Superti Fabiana , Franco Marina , Melucci-Vigo Gianna , Cafaro Aurelio , Goletti Delia , Sgadari Cecilia , Butto’ Stefano , Leone Patrizia , Leone Pasqualina , Chiozzini Chiara , Barresi Caterina , Tinari Antonella , Bonaccorsi Angela , Capobianchi Maria R. , Giuliani Massimo , di Carlo Aldo , Andreoni Massimo , Rezza Giovanni , Ensoli Barbara
Published: December 2019

Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi’s sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-α), we analyzed the effects of IFN-α or anti-IFN-α antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-α inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8nut-1and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-α inhibited virus production and/or release from BCBL-1 cells. Inhibition ofnut-1and kaposin genes by IFN-α was also observed in BC-1 cells induced withn-butyrate. Conversely, the addition of anti-IFN-α Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-α Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-α reduced the HHV-8 load in PBMC. Thus, IFN-α appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-α may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.
Cellular Tropism and Viral Interleukin-6 Expression Distinguish Human Herpesvirus 8 Involvement in Kaposi’s Sarcoma, Primary Effusion Lymphoma, and Multicentric Castleman’s Disease
Authors: Staskus Katherine A. , Sun Ren , Miller George , Racz Paul , Jaslowski Anthony , Metroka Craig , Brett-Smith Helena , Haase Ashley T.
Published: December 2019

Human herpesvirus 8 (HHV-8) infection has been implicated in the etiology of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD), three diseases that frequently develop in immunocompromised, human immunodeficiency virus-positive individuals. One hypothesis that would account for different pathological manifestations of infection by the same virus is that viral genes are differentially expressed in heterogeneous cell types. To test this hypothesis, we analyzed the localization and levels of expression of two viral genes expressed in latent and lytic infections and the viral homologue of interleukin-6 (vIL-6). We show that PEL parallels KS in the pattern of latent and lytic cycle viral gene expression but that the predominant infected cell type is a B cell. We also show that MCD differs from KS not only in the infected cell type (B-cell and T-cell lineage) but also in the pattern of viral gene expression. Only a few cells in the lesion are infected and all of these cells express lytic-cycle genes. Of possibly greater significance is the fact that in a comparison of KS, PEL, and MCD, we found dramatic differences in the levels of expression of vIL-6. Interleukin-6 is a B-cell growth and differentiation factor whose altered expression has been linked to plasma cell abnormalities, as well as myeloid and lymphoid malignancies. Our findings support the hypothesis that HHV-8 plays an important role in the pathogenesis of PEL and MCD, in which vIL-6 acts as an autocrine or paracrine factor in the lymphoproliferative processes common to both.
The Amino-Terminal Region of Vpr from Human Immunodeficiency Virus Type 1 Forms Ion Channels and Kills Neurons
Authors: Piller S. C. , Ewart G. D. , Jans D. A. , Gage P. W. , Cox G. B.
Published: December 2019

We have previously reported that the accessory protein Vpr from human immunodeficiency virus type 1 forms cation-selective ion channels in planar lipid bilayers and is able to depolarize intact cultured neurons by causing an inward sodium current, resulting in cell death. In this study, we used site-directed mutagenesis and synthetic peptides to identify the structural regions responsible for the above functions. Mutations in the N-terminal region of Vpr were found to affect channel activity, whereas this activity was not affected by mutations in the hydrophobic region of Vpr (amino acids 53 to 71). Analysis of mutants containing changes in the basic C terminus confirmed previous results that this region, although not necessary for ion channel function, was responsible for the observed rectification of wild-type Vpr currents. A peptide comprising the first 40 N-terminal amino acids of Vpr (N40) was found to be sufficient to form ion channels similar to those caused by wild-type Vpr in planar lipid bilayers. Furthermore, N40 was able to cause depolarization of the plasmalemma and cell death in cultured hippocampal neurons with a time course similar to that seen with wild-type Vpr, supporting the idea that this region is responsible for Vpr ion channel function and cytotoxic effects. Since Vpr is found in the serum and cerebrospinal fluids of AIDS patients, these results may have significance for AIDS pathology.
Gag Protein Epitopes Recognized by CD4+T-Helper Lymphocytes from Equine Infectious Anemia Virus-Infected Carrier Horses
Authors: Lonning S. M. , Zhang W. , McGuire T. C.
Published: December 2019

Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAVgaggene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV.
Quantitative Analysis of the Acute and Long-Term CD4+T-Cell Response to a Persistent Gammaherpesvirus
Authors: Christensen Jan P. , Doherty Peter C.
Published: December 2019

The murine gammaherpesvirus 68 (MHV-68) replicates in respiratory epithelial cells, where it establishes a persistent, latent infection limited predominantly to B lymphocytes. The virus-specific CD4+T-cell response in C57BL/6 mice challenged intranasally with MHV-68 is detected first in the mediastinal lymph nodes and then in the cervical lymph nodes and the spleen. The numbers of MHV-68-specific CD4+T cells generated in congenic mice homozygous for disruption of the β2-microglobulin gene tended to be higher, indicating that the absence of the CD8+set in this group resulted in a compensatory response. The peak frequency within the splenic CD4+T-cell population may reach 1:50 in the acute response; it then drops to 1:400 to 1:500 within 4 months and stays at that level in the very long term. Sorting for L-selectin (CD62L) expression established that all virus-specific CD4+T cells were initially CD62Llow, with >80% maintaining that phenotype for the next 14 months. The overall conclusion is that MHV-68-specific CD4+T cells remain activated (CD62Llow) and at a stable frequency in the face of persistent infection.
Adeno-Associated Virus (AAV) Type 5 Rep Protein Cleaves a Unique Terminal Resolution Site Compared with Other AAV Serotypes
Authors: Chiorini John A. , Afione Sandra , Kotin Robert M.
Published: December 2019

Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) in
trans
, and inverted terminal repeat (ITR) sequences in
cis
. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro replication assays indicated that the block occurs at the level of replication instead of at viral assembly. AAV2 and AAV5 Rep binding activities demonstrate similar affinities for either an AAV2 or AAV5 ITR; however, comparison of terminal resolution site (TRS) endonuclease activities showed a difference in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a type 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficiently. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG TGGC) which is absent from the ITRs of other AAV serotypes. Comparison of the TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage site but little homology upstream.