scholarly journals Control of Cytoplasmic Maturation Events by Cytomegalovirus Tegument Protein pp150

2008 ◽  
Vol 82 (19) ◽  
pp. 9433-9444 ◽  
Author(s):  
Ritesh Tandon ◽  
Edward S. Mocarski
Keyword(s):  
Cellular Localization ◽  
Cytoplasmic Inclusion ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Virion Assembly ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
Assembly Compartment

ABSTRACT Cytomegalovirus replication depends upon a betaherpesvirus-conserved 150-kDa tegument phosphoprotein (pp150; encoded by UL32) that supports the final steps in virion maturation at cytoplasmic assembly compartments. Amino acid substitutions were introduced into conserved region 1 (CR1) and CR2 of pp150, affecting a region that may interact with nucleocapsids. Two independent CR2 point mutants (N201A and G207A) failed to support viral replication in evaluations by a transient complementation assay or after reconstruction into recombinant viruses. An assembly compartment-like cytoplasmic inclusion developed in UL32 mutant virus-infected cells that was similar to that of wild-type virus-infected cells. The cellular localization of the trans-Golgi marker Golgin-97 suggested differences in the organization of the assembly compartment compared to that of wild-type virus-infected cells. Replication-defective CR2 point mutants exhibited the same phenotype as that of a virus carrying a complete deletion of the UL32 open reading frame in these assays. Electron micrographs of fibroblasts at 3 or 5 days postinfection with a deletion mutant (ΔUL32) grown on UL32-complementing cells showed a similar number and morphology of capsids in the nucleus, but the cytoplasmic region associated with virion assembly appeared highly vesiculated and contained few recognizable nucleocapsids or complete virus particles. These data demonstrate that the principle role of pp150 is to retain nucleocapsid organization through secondary envelopment at the assembly compartment.

scholarly journals Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection

2001 ◽  
Vol 75 (20) ◽  
pp. 9623-9632 ◽  
Author(s):  
Irmgard Pult ◽  
Nathan Abbott ◽  
Yong-Yuan Zhang ◽  
Jesse Summers
Keyword(s):  
Nucleotide Substitution ◽  
Acute Liver Injury ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Single Nucleotide ◽  
Reading Frame ◽  
Duck Hepatitis ◽  
B Virus ◽  
Infected Cells

ABSTRACT In this study, we measured the frequency of revertants of a cytopathic strain of the duck hepatitis B virus that bears a single nucleotide substitution in the pre-S envelope protein open reading frame, resulting in the amino acid substitution G133E. Cytopathic virus mixed with known amounts of a genetically marked wild-type virus was injected into ducklings. Virus outgrowth was accompanied by a coselection of wild-type and spontaneous revertants during recovery of the ducklings from the acute liver injury caused by death of the G133E-infected cells. The frequency of individual revertants in the selected noncytopathic virus population was estimated by determining the ratio of each revertant to the wild-type virus. Spontaneous revertants were found to be present at frequencies of 1 × 10−5 to 6 × 10−5 per G133E genome inoculated. A mathematical model was used to estimate that the mutation rate was 0.8 × 10−5 to 4.5 × 10−5per nucleotide per generation.

scholarly journals In Vivo Dynamics of Reporter Flaviviridae Viruses

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Tomokazu Tamura ◽  
Manabu Igarashi ◽  
Bazarragchaa Enkhbold ◽  
Tatsuya Suzuki ◽  
Masatoshi Okamatsu ◽  
...  
Keyword(s):  
Luciferase Activity ◽  
Viral Proteins ◽  
Solvent Accessible Surface Area ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Diarrhea Virus ◽  
Foreign Genes

ABSTRACT Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo. Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro. The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals. IMPORTANCE In vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo. Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

scholarly journals The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection

2019 ◽  
Vol 32 (7) ◽  
pp. 865-875 ◽  
Author(s):  
Kegui Chen ◽  
Behnam Khatabi ◽  
Vincent N. Fondong
Keyword(s):  
Plant Viruses ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Type Virus ◽  
Wild Type ◽  
Knockout Mutant ◽  
East African ◽  
Reading Frame ◽  
Cell Membrane Binding

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

scholarly journals Interwoven Roles of Cyclin D3 and cdk4 Recruited by ICP0 and ICP4 in the Expression of Herpes Simplex Virus Genes

2010 ◽  
Vol 84 (19) ◽  
pp. 9709-9717 ◽  
Author(s):  
Maria Kalamvoki ◽  
Bernard Roizman
Keyword(s):  
Cyclin D3 ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Nuclear Retention ◽  
Enhanced Expression ◽  
Infected Cells ◽  
Cytoplasmic Accumulation ◽  
Simplex Virus ◽  
Cdk4 Inhibitor

ABSTRACT Elsewhere this laboratory reported that (i) ICP0 interacts with cyclin D3 but not D1 or D2. The 3 cyclins independently partially rescue ΔICP0 mutants. (ii) Interaction with cyclin D3 is required for the switch from nuclear to cytoplasmic accumulation of ICP0. (iii) In infected cells cdk4 is activated whereas cdk2 is not. Inhibition of cdk4 results in nuclear retention of ICP0. Overexpression of cyclin D3 reverses the effect of the inhibitor. Here we report the following. (i) cdk4 interacts with ICP0, ICP4, and possibly with ICP8. This interaction is required to recruit cdk4 initially to ND10 and later to the viral replication compartments. (ii) cdk4 inhibitor I reduced or delayed the transcription and ultimately translation of mRNAs of ICP4, ICP27, or ICP8 and to a lesser extent that of the ICP0 gene in wild-type virus-infected cells. (iii) Overexpression of cyclin D3 resulted in a more rapid transcription of these genes. In the presence of inhibitor, the rates of accumulation of the products of these genes resemble those of wild-type virus in the absence of inhibitor. (iv) Overexpression of cyclin D3 also results in mobilization of cdk6 in nuclei of infected cells. We conclude that ICP0 encodes a function that enhances the recruitment of cyclin D3 to ND10 structures to activate cdk4 and that ICP0 along with other viral proteins recruits cdk4 to ND10 structures and ultimately to replication compartments for enhanced expression of viral genes and viral DNA synthesis.

scholarly journals Herpes Simplex Virus 1 Induces Cytoplasmic Accumulation of TIA-1/TIAR and both Synthesis and Cytoplasmic Accumulation of Tristetraprolin, Two Cellular Proteins That Bind and Destabilize AU-Rich RNAs

2004 ◽  
Vol 78 (16) ◽  
pp. 8582-8592 ◽  
Author(s):  
Audrey Esclatine ◽  
Brunella Taddeo ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Cytoplasmic Accumulation ◽  
Simplex Virus

ABSTRACT Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the UL41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3′-to-5′ degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking UL41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking UL41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.

scholarly journals Human Cytomegalovirus pUS24 Is a Virion Protein That Functions Very Early in the Replication Cycle

2006 ◽  
Vol 80 (17) ◽  
pp. 8371-8378 ◽  
Author(s):  
Xuyan Feng ◽  
Jörg Schröer ◽  
Dong Yu ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Mutant Virus ◽  
Replication Cycle ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Protein ◽  
Viral Dna ◽  
Infected Cells

ABSTRACT We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.

Evaluation of Influenza A/Hong Kong/123/77 (H1N1) ts -1A2 and Cold-Adapted Recombinant Viruses in Seronegative Adult Volunteers

1980 ◽  
Vol 29 (2) ◽  
pp. 348-355 ◽  
Author(s):  
Brian R. Murphy ◽  
Margret B. Rennels ◽  
R. Gordon Douglas ◽  
Robert F. Betts ◽  
Robert B. Couch ◽  
...  
Keyword(s):  
Hemagglutination Inhibition ◽  
Influenza A ◽  
Wild Type Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Response ◽  
Type Virus ◽  
Wild Type ◽  
Cold Adapted ◽  
Recombinant Viruses ◽  
Inhibition Response

Two attenuated influenza A donor viruses, the A/Udorn/72 ts -1A2 and the A/Ann Arbor/6/60 cold-adapted ( ca ) viruses, are being evaluated for their ability to reproducibly attenuate each new variant of influenza A virus to a specific and desired level by the transfer of one or more attenuating genes. Each of these donor viruses has been able to attenuate influenza A viruses belonging to the H3N2 subtype by the transfer of one or more attenuating genes. To determine whether these two donor viruses could attenuate a wild-type virus that belonged to a different influenza A subtype, ts -1A2 and ca recombinants of a wild-type virus representative of the A/USSR/77 (H1N1) Russian influenza strain were prepared and evaluated in adult doubly seronegative volunteers at several doses. The recombinants derived from both donor viruses were attenuated for the doubly seronegative adults. Less than 5% of infected vaccinees developed a febrile or systemic reaction, whereas five of six recipients of wild-type virus developed such a response. The 50% human infectious dose (HID 50 ) for each recombinant was approximately 10 5.0 50% tissue culture infective doses. The virus shed by the ts -1A2 and ca vaccinees retained the ts or ca phenotype, or both. This occurred despite replication of the recombinant viruses for up to 9 days. No evidence for transmission of the ca or ts -1A2 recombinant virus to controls was observed. A serum hemagglutination inhibition response was detected in less than 50% of the infected vaccinees. However, with the more sensitive enzyme-linked immunosorbent assay, a serological response was detected in 100% of the ca vaccinees given 300 HID 50 and approximately 70% of ca or ts vaccinees who received 10 to 32 HID 50 of virus. These results indicate that the recombinants derived from both donor viruses were satisfactorily attenuated and were stable genetically after replication in doubly seronegative adults although they induced a lower serum hemagglutination inhibition response than that found previously for H3N2 ts and ca recombinants.

scholarly journals Requirements for the Nuclear-Cytoplasmic Translocation of Infected-Cell Protein 0 of Herpes Simplex Virus 1

2001 ◽  
Vol 75 (8) ◽  
pp. 3832-3840 ◽  
Author(s):  
Pascal Lopez ◽  
Charles Van Sant ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Regulatory Protein ◽  
Wild Type Virus ◽  
Cell Protein ◽  
Type Virus ◽  
Wild Type ◽  
Infected Cells ◽  
Infected Cell Protein ◽  
Simplex Virus

ABSTRACT Earlier studies have shown that wild-type infected-cell protein 0 (ICP0), a key herpes simplex virus regulatory protein, translocates from the nucleus to the cytoplasm of human embryonic lung (HEL) fibroblasts within several hours after infection (Y. Kawaguchi, R. Bruni, and B. Roizman, J. Virol. 71:1019–1024, 1997). Translocation of ICP0 was also observed in cells infected with thed120 mutant, in which both copies of the gene encoding ICP4, the major regulatory protein, had been deleted (V. Galvan, R. Brandimarti, J. Munger, and B. Roizman, J. Virol. 74:1931–1938, 2000). Furthermore, a mutant (R7914) carrying the D199A substitution in ICP0 does not bind or stabilize cyclin D3 and is retained in the nucleus (C. Van Sant, P. Lopez, S. J. Advani, and B. Roizman, J. Virol. 75:1888–1898, 2001). Studies designed to elucidate the requirements for the translocation of ICP0 between cellular compartments revealed the following. (i) Translocation of ICP0 to the cytoplasm in productive infection maps to the D199 amino acid, inasmuch as wild-type ICP0 delivered in trans to cells infected with an ICP0 null mutant was translocated to the cytoplasm whereas the D199A-substituted mutant ICP0 was not. (ii) Translocation of wild-type ICP0 requires a function expressed late in infection, inasmuch as phosphonoacetate blocked the translocation of ICP0 in wild-type virus-infected cells but not in d120 mutant-infected cells. Moreover, whereas in d120 mutant-infected cells ICP0 was translocated rapidly from the cytoplasm to the nucleus at approximately 5 h after infection, the translocation of ICP0 in wild-type virus-infected cells extended from 5 to at least 9 h after infection. (iii) In wild-type virus-infected cells, the MG132 proteasomal inhibitor blocked the translocation of ICP0 to the cytoplasm early in infection, but when added late in infection, it caused ICP0 to be relocated back to the nucleus from the cytoplasm. (iv) MG132 blocked the translocation of ICP0 in d120 mutant-infected cells early in infection but had no effect on the ICP0 aggregated in vesicle-like structures late in infection. However, ind120 mutant-infected cells treated with MG132 at late times, proteasomes formed a shell-like structure around the aggregated ICP0. These structures were not seen in wild-type virus or R7914 mutant-infected cells. The results indicate the following. (i) In the absence of β or γ protein synthesis, ICP0 dynamically associates with proteasomes and is translocated to the cytoplasm. (ii) In cells productively infected beyond α gene expression, ICP0 is retained in the nucleus until after the onset of viral DNA synthesis and the synthesis of γ2 proteins. (iii) Late in infection, ICP0 is actively sequestered in the cytoplasm by a process mediated by proteasomes, inasmuch as interference with proteasomal function causes rapid relocation of ICP0 to the nucleus.

scholarly journals Genetically Engineered Vesicular Stomatitis Virus in Gene Therapy: Application for Treatment of Malignant Disease

2002 ◽  
Vol 76 (2) ◽  
pp. 895-904 ◽  
Author(s):  
Marilyn Fernandez ◽  
Mercedes Porosnicu ◽  
Dubravka Markovic ◽  
Glen N. Barber
Keyword(s):  
Gene Therapy ◽  
Vesicular Stomatitis Virus ◽  
Mammary Adenocarcinoma ◽  
Wild Type Virus ◽  
Cytotoxic T Cell ◽  
Type Virus ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Vesicular Stomatitis ◽  
Oncolytic Activity

ABSTRACT We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.

scholarly journals Translocation and Colocalization of ICP4 and ICP0 in Cells Infected with Herpes Simplex Virus 1 Mutants Lacking Glycoprotein E, Glycoprotein I, or the Virion Host Shutoff Product of the UL41 Gene

2007 ◽  
Vol 82 (4) ◽  
pp. 1701-1713 ◽  
Author(s):  
Maria Kalamvoki ◽  
Jianguo Qu ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Glycoprotein E ◽  
Glycoprotein I ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT In wild-type herpes simplex virus 1-infected cells, the major regulatory protein ICP4 resides in the nucleus whereas ICP0 becomes dynamically associated with proteasomes and late in infection is translocated and dispersed in the cytoplasm. Inhibition of proteasomal function results in retention or transport of ICP0 to the nucleus. We report that in cells infected with mutants lacking glycoprotein E (gE), glycoprotein I (gI), or the product of the UL41 gene, both ICP4 and ICP0 are translocated to the cytoplasm and coaggregate in small dense structures that, in the presence of proteasomal inhibitor MG132, also contain proteasomal components. Gold particle-conjugated antibody to ICP0 reacted in thin sections with dense protein aggregates in the cytoplasm of mutant virus-infected cells. Similar aggregates were present in the nuclei but not in the cytoplasm of wild-type virus-infected cells. Exposure of cells early in infection to MG132 does not result in retention of ICP0 as in wild-type virus-infected cells. The results suggest that the retention of ICP4 and ICP0 in the nucleus is a dynamic process that involves the function of other viral proteins that may include the Fc receptor formed by the gE/gI complex and is not merely the consequence of expression of a nuclear localization signal. It is noteworthy that in ΔUL41-infected cells gE is retained in the trans-Golgi network and is not widely dispersed in cellular membranes.

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