Downregulation of autophagy by herpesvirus Bcl-2 homologs

The critical role of the cellular autophagy pathway in viral infection and pathogenesis has become increasingly apparent. Mounting evidences suggest that viruses have developed different strategies to meticulously modulate intracellular autophagy for their own benefits, thereby either promoting efficient viral replication or facilitating viral persistence. While our understanding of these strategies is still in its incipient stage, recent advances demonstrate that gamma herpesvirus Bcl-2 homolog (vBcl-2), which protects virus-infected cells from apoptosis, also suppresses cellular autophagy pathway through its direct interaction with the autophagy protein Beclin1. Interestingly, vBcl-2 has evolved to harbor the enhanced anti-autophagic activity compared to its host counterpart, suggesting an important role of cellular autophagy in response to viral infection and virus-associated pathogenesis. Thus, a detailed study of vBcl-2-mediated regulation of autophagy signal transduction pathway may lead to a better understanding of not only how virus escapes from host innate immunity but also how autophagy regulates viral infection and environmental stresses.     

Mammalian diseases of phosphatidylinositol transfer proteins and their homologs

Inositol and phosphoinositide signaling pathways represent major regulatory systems in eukaryotes. The physiological importance of these pathways is amply demonstrated by the variety of diseases that involve derangements in individual steps in inositide and phosphoinositide production and degradation. These diseases include numerous cancers, lipodystrophies and neurological syndromes. Phosphatidylinositol transfer proteins (PITPs) are emerging as fascinating regulators of phosphoinositide metabolism. Recent advances identify PITPs (and PITP-like proteins) to be coincidence detectors, which spatially and temporally coordinate the activities of diverse aspects of the cellular lipid metabolome with phosphoinositide signaling. These insights are providing new ideas regarding mechanisms of inherited mammalian diseases associated with derangements in the activities of PITPs and PITP-like proteins.     

Plant virus satellite RNAs and their role in engineering resistance to virus diseases

The mechanism of satellite RNA (Sat-RNA) mediated attenuation of virus disease was suggested to be the result of competition between satellite RNAs and their helper viral RNAs for replication. Subcellular distribution of viral coat protein in cucumber mosaic virus (CMV) infected leaf tissues shows that the presence of Sat-RNAs interferes with the movement of CMV coat protein into chloroplants. As a strategy for controlling virus diseases, the expression of Sat-RNAs in transgenic plants confers some extent of resistance that may not be strong enough to protect the plants from natural virus infections. Transgenic plants expressing both the Sat-RNA and the coat protein of CMV exhibit enhanced resistance to the virus.     

病毒工厂：病毒复制的关键场所

多种病毒的复制和组装过程需要在被称为"病毒工厂"的特殊结构内完成。随着研究水平的不断提高,研究者已经在一定程度上揭示了病毒工厂的形成过程及结构。病毒入侵细胞后,能够招募细胞和病毒成分从而形成病毒组装和成熟的场所,细胞膜结构和细胞骨架能够参与该结构的形成,且部分病毒形成的病毒工厂还需线粒体提供能量。除上述特征外,病毒工厂的结构及形态会随病毒复制阶段的不同而不断变化。本文将对病毒工厂的结构、细胞器的招募、病毒工厂结构的变化及大分子物质的运输进行综述。     

Antiapoptotic herpesvirus Bcl-2 homologs escape caspase-mediated conversion to proapoptotic proteins

The antiapoptotic Bcl-2 and Bcl-x(L) proteins of mammals are converted into potent proapoptotic factors when they are cleaved by caspases, a family of apoptosis-inducing proteases (E. H.-Y. Cheng, D. G. Kirsch, R. J. Clem, R. Ravi, M. B. Kastan, A. Bedi, K. Ueno, and J. M. Hardwick, Science 278:1966-1968, 1997; R. J. Clem, E. H.-Y. Cheng, C. L. Karp, D. G. Kirsch, K. Ueno, A. Takahashi, M. B. Kastan, D. E. Griffin, W. C. Earnshaw, M. A. Veliuona, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 95:554-559, 1998). Gamma herpesviruses also encode homologs of the Bcl-2 family. All tested herpesvirus Bcl-2 homologs possess antiapoptotic activity, including the more distantly related homologs encoded by murine gammaherpesvirus 68 (gammaHV68) and bovine herpesvirus 4 (BHV4), as described here. To determine if viral Bcl-2 proteins can be converted into death factors, similar to their cellular counterparts, five herpesvirus Bcl-2 homologs from five different viruses were tested for their susceptibility to caspases. Only the viral Bcl-2 protein encoded by gammaHV68 was susceptible to caspase digestion. However, unlike the caspase cleavage products of cellular Bcl-2, Bcl-x(L), and Bid, which are potent inducers of apoptosis, the cleavage product of gammaHV68 Bcl-2 lacked proapoptotic activity. KSBcl-2, encoded by the Kaposi's sarcoma-associated herpesvirus, was the only viral Bcl-2 homolog that was capable of killing cells when expressed as an N-terminal truncation. However, because KSBcl-2 was not cleavable by caspases, the latent proapoptotic activity of KSBcl-2 apparently cannot be released. The Bcl-2 homologs encoded by herpesvirus saimiri, Epstein-Barr virus, and BHV4 were not cleaved by apoptotic cell extracts and did not possess latent proapoptotic activities. Thus, herpesvirus Bcl-2 homologs escape negative regulation by retaining their antiapoptotic activities and/or failing to be converted into proapoptotic proteins by caspases during programmed cell death.     

Viral and cellular determinants of hepatitis C virus RNA replication in cell culture

Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i). mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii). mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.     

New insights in the role of Bcl-2 Bcl-2 and the endoplasmic reticulum

The oncogenic protein Bcl-2 which is expressed in membranes of different subcellular organelles protects cells from apoptosis induced by endogenic stimuli. Most of the results published so far emphasise the importance of Bcl-2 at the mitochondria. Several recent observations suggest a role of Bcl-2 at the endoplasmic reticulum (ER). Bcl-2 located at the ER was shown to interfere with apoptosis induction by Bax, ceramides, ionising radiation, serum withdrawal and c-myc expression. Although the detailed functions of Bcl-2 at the ER remain elusive, several speculative mechanisms may be supposed. For instance, Bcl-2 at the ER may regulate calcium fluxes between the ER and the mitochondria. In addition, Bcl-2 is able to interact with the endoplasmic protein Bap31 thus avoiding caspase activation at the ER. Bcl-2 may also abrogate the function of ER located pro-apoptotic Bcl-2 like proteins by heterodimerization. Current data on the function of Bcl-2 at the ER, its role for the modulation of calcium fluxes and its influence on caspase activation at the ER are reviewed.     

Survival activity of Bcl-2 homologs Bcl-w and A1 only partially correlates with their ability to bind pro-apoptotic family members.

Certain Bcl-2 family members promote cell survival, whereas others promote apoptosis. To explore further how heterodimerization of opposing members affects survival activity, we have compared the abilities of the anti-apoptotic Bcl-w and A1 to bind to the pro-apoptotic Bax, Bak, Bad and Bik and to protect cells from their cytotoxic action. Bcl-w co-immunoprecipitated from cell lysates with Bax, Bak, Bad and Bik, but A1 bound only Bak and Bik. Mutation of A1 at a highly conserved glycine within the BH1 domain prevented binding, but the comparable Bcl-w mutant still bound Bak, Bad and Bik, indicating that the glycine is not essential for all heterodimerization. Bcl-w and A1 protected against apoptosis induced by over-expression of Bax or Bad but not that induced by Bak or Bik. With several gene pairs, binding and protection were discordant. The results may reflect critical threshold affinities but also suggest that certain pro-apoptotic proteins may also contribute to apoptosis by a mechanism independent of binding pro-survival proteins.     

SARS-CoV-2 Mutations and their Viral Variants

Mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occur spontaneously during replication. Thousands of mutations have accumulated and continue to since the emergence of the virus. As novel mutations continue appearing at the scene, naturally, new variants are increasingly observed.Since the first occurrence of the SARS-CoV-2 infection, a wide variety of drug compounds affecting the binding sites of the virus have begun to be studied. As the drug and vaccine trials are continuing, it is of utmost importance to take into consideration the SARS-CoV-2 mutations and their respective frequencies since these data could lead the way to multi-drug combinations. The lack of effective therapeutic and preventive strategies against human coronaviruses (hCoVs) necessitates research that is of interest to the clinical applications.The reason why the mutations in glycoprotein S lead to vaccine escape is related to the location of the mutation and the affinity of the protein. At the same time, it can be said that variations should occur in areas such as the receptor-binding domain (RBD), and vaccines and antiviral drugs should be formulated by targeting more than one viral protein.In this review, a literature survey in the scope of the increasing SARS-CoV-2 mutations and the viral variations is conducted. In the light of current knowledge, the various disguises of the mutant SARS-CoV-2 forms and their apparent differences from the original strain are examined as they could possibly aid in finding the most appropriate therapeutic approaches.     

Viral protein R of human immunodeficiency virus types 1 and 2 is dispensable for replication and cytopathogenicity in lymphoid cells.

Viral protein R (VPR) is conserved in human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). To assess its function, we have constructed mutations within the vpr coding regions of HIV-1 and HIV-2 predicted to express truncated VPR products. Infectious virus was produced by each proviral clone and showed similar replication kinetics and cytopathogenicity when compared with the corresponding parental proviral clone.     

Dispensable role of the human immunodeficiency virus type 2 Vpx protein in viral replication.

Human immunodeficiency virus type 2 (HIV-2) is similar in genetic organization to HIV-1 but contains a unique gene (vpx) that encodes a 16-kDa protein. A replication-competent molecular clone of HIV-2 (HIV-2sbl/isy) that infects human primary cells in vitro and rhesus monkeys was used to generate three mutations in the vpx gene. In the first mutant, the vpx open reading frame was truncated at amino acid 20; the second mutant was tailored to eliminate the proline-rich carboxyl terminus of the protein; and the third mutant was obtained by addition of four amino acids (KDEL) to the carboxyl terminus of the protein to provide a retention signal in the endoplasmic reticulum. The viral infection kinetics of the three mutant viruses and isogeneic HIV-2sbl/isy in the SupT1 cell line were similar. Slight impairment in the early phases of viral replication was observed during infection of primary human peripheral blood mononuclear cells with the vpx mutant viruses. All of the vpx mutant viruses readily infected macrophages, indicating that vpx expression is dispensable for HIV-2 infection and replication in human macrophages.     

Epstein-barr virus replication studies and their application to vector design

Vectors containing elements of the Epstein-Barr virus genome are used primarily to maintain cloned DNA inserts as plasmids in mammalian cells. However, Epstein-Bar-virus-based vectors have also been valuable tools in the hands of those studying the life cycle of Epstein-Barr virus. In this article, we discuss those characteristics of Epstein-Barr virus and its life cycle that have been used in vector construction and describe methods that are particularly applicable to the use of Epstein-Barr-virus-based vectors.     

Merosin (laminin-2/4)-driven survival signaling: complex modulations of Bcl-2 homologs

We have shown previously that the promotion of myofiber survival by the basement membrane component merosin (laminin-2 [alpha2beta1gamma1]/laminin-4 [alpha2beta2gamma1]) is dependent on the activity of the tyrosine kinase Fyn, whereas myofiber anoikis induced by merosin deficiency is dependent on the stress-activated protein kinase p38alpha. To further understand such merosin-driven survival signaling, we analyzed the expression of five Bcl-2 homologs (Bcl-2, Bcl-X(L), Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1) in normal and merosin-deficient myotubes, with or without pharmacological inhibitors for Fyn and p38. Herein, we report that (1) merosin deficiency induces anoikis and causes decreased Bcl-2, Bcl-X(L), and Bag-1 levels, increased Bax and Bak levels, and decreased Bad phosphorylation; (2) Bcl-2, Bcl-X(L), Bag-1, and Bad phosphorylation are also decreased in anoikis-dying, Fyn-inhibited myotubes; (3) the inhibition of p38alpha in Fyn-inhibited and/or merosin-deficient myotubes protects against anoikis and increases Bcl-2 levels above normal, in addition to restoring Bad phosphorylation and Bag-1 levels to normal; (4) the overexpression of merosin in deficient myotubes also rescues from anoikis and increases Bcl-2 levels and Bad phosphorylation above normal, in addition to restoring Bcl-X(L), Bag-1, Bax, and Bak levels to normal; and (5) Bcl-2 overexpression is sufficient to rescue merosin-deficient myotubes from anoikis, even though the expression/phosphorylation levels of the other homologs analyzed are not restored to normal. These results indicate that merosin-driven myofiber survival signaling affects complex, differential modulations of individual Bcl-2 homologs. These further suggest that Bcl-2 can play a major role in suppressing myofiber anoikis.     

Viral evolution and interferon resistance of hepatitis C virus RNA replication in a cell culture model

Hepatitis C virus (HCV) replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. We evaluated HCV-IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV type 1b subgenomic RNA replicon that differed at only two sites within the NS5A-encoding region. A replicon with a K insertion at HCV codon 2040 replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an L2198S point mutation replicated poorly and triggered a cellular response characterized by IFN-beta production and low-level IFN-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high-passage (HP) variant with six additional point mutations throughout the HCV protein-encoding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1,000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-alpha treatment and was associated with virus-directed reduction in host cell expression of ISG56, an antagonist of HCV RNA translation. Accordingly, the HP RNA was retained within polyribosome complexes in vivo that were refractory to IFN-induced disassembly. These results identify ISG56 as a translational control effector of the host response to HCV and provide direct evidence to link this response to viral sequence evolution, ISG regulation, and selection of the IFN-resistant viral phenotype.