Roles of the three major phosphorylation sites of hepatitis B virus core protein in viral replication.

Hepatitis B virus (HBV) core protein is a phosphoprotein. Its three major phosphorylation sites have been identified at the serine residues located at amino acids 157, 164, and 172. In order to investigate the role of core protein phosphorylation in HBV replication, these three serine residues were mutated to alanine to mimic nonphosphorylated serine or to glutamic acid to mimic phosphoserine. The nonphosphorylated core protein analog did not package the HBV pregenomic RNA, and the phosphorylated analog packaged the pregenomic RNA but failed to support viral DNA replication. These results indicate that the core protein phosphorylation may be important for pregenomic RNA packaging and that its dephosphorylation may be important for viral DNA replication. The individual roles of these three major phosphorylation sites in HBV replication were further investigated by being mutated to alanine in different combinations. The results showed that the serine residue at amino acid 157 was not essential for pregenomic RNA packaging, whereas the serine residues at amino acids 164 and 172 were more important. Furthermore, the serine residue at amino acid 157 was not essential for viral DNA replication or viral maturation.     

Involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin A.

Brefeldin A (BFA) is a macrolide antibiotic that has multiple targets in vesicular transport and blocks membrane traffic between the cis- and trans-Golgi compartments, leading to the disruption of the trans-Golgi apparatus (for a review see Pelham, 1991, Cell 67, 449-451). Consequently, BFA interferes with the maturation of viral glycoproteins and suppresses the formation of infectious viruses that contain a lipid envelope. We report that this antibiotic strongly inhibits poliovirus replication even though this virus lacks a lipid envelope and does not encode any glycoproteins. Addition of BFA from the beginning of poliovirus infection blocks the synthesis of late proteins but has no effect on p220 cleavage, indicating that the input viral RNA is translated to produce active 2Apro. The presence of BFA at later times has no effect on poliovirus protein synthesis, indicating that this step is not a direct target for the antibiotic. Indeed, the target of BFA is viral RNA synthesis, because addition of the antibiotic at any time after poliovirus infection drastically reduces the incorporation of labeled uridine into poliovirus RNA. Both plus- and minus-stranded RNA syntheses are diminished when BFA is present from the beginning of infection, but plus-stranded RNA synthesis is more affected when the inhibitor is added at later times. The replication of poliovirus RNA takes place in close association with membrane vesicles that fill the cytoplasm of the infected cells. Little is known about the origin and function of these vesicles that form part of the viral replication complexes. Our findings suggest that the replication of poliovirus genomes may require the maturation of membranous vesicles from a vesicular compartment that is affected by BFA. The effects of BFA on late protein synthesis by other animal viruses varies according to the virus species examined. Among picornaviruses, rhinoviruses are sensitive to the antibiotic, whereas encephalomyocarditis virus is resistant. A negative-stranded RNA virus such as vesicular stomatitis is blocked by BFA, whereas vaccinia virus, a cytoplasmic DNA virus, is resistant.     

Localization of hepatitis B virus core protein and viral DNA at the nuclear membrane

An early step in the replication of the hepatitis B virus (HBV) genome is the transport of the viral DNA into the nucleus of the infected cell. So far only little is known about the events and mechanisms at the nuclear membrane required for entry of the viral genome into the nucleus. Using a hepatoblastoma cell line that constitutively produces hepatitis B virions and in so doing displays intracellular viral amplification, we showed that nonparticulated HBV core protein is associated with nuclear membrane pore complexes. Additionally, viral DNA has been detected firmly attached to the nuclear membrane. Small amounts of viral core protein, as well as viral DNA, were detectable within the cell nucleus. However, core particles could not be shown at the nuclear membrane or within the nuclei of these cells. Our observations on localization of HBV DNA and core protein at the nuclear membrane thus provide a suggestion for further examinations of the transfer of the viral genome from the cytoplasm into the nucleus of the infected cell.     

The expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis

Abstract

The endoplasmic reticulum (ER) is a cellular membrane organelle that plays important roles in virus replication and maturation. Accumulating evidence indicates that virus infection often disturbs ER homeostasis and leads to ER stress, which is associated with a variety of prevalent diseases. To cope with the deleterious effects of virus-induced ER stress, cells activate critical signaling pathways including the unfolded protein response (UPR) and intrinsic mitochondrial apoptosis, which have complex effects on virus replication and pathogenesis. In this review, we present a comprehensive summary of recent research in this field, which revealed that about 36 viruses trigger ER stress and differentially activate ER stress-related signaling pathways. We also highlight the strategies evolved by viruses to modulate ER stress-related signaling networks including immune responses in order to ensure their survival and pathogenesis. Together, the knowledge gained from this field will shed light on unveiling the mechanisms of virus replication and pathogenesis and provide insight for future research as well as antiviral development.     

Palmitoylation of CM2 is dispensable to influenza C virus replication

CM2 is the second membrane protein of influenza C virus. The significance of the posttranslational modifications of CM2 remains to be clarified in the context of viral replication, although the positions of the modified amino acids on CM2 have been determined. In the present study, using reverse genetics we generated rCM2-C65A, a recombinant influenza C virus lacking CM2 palmitoylation site, in which cysteine at residue 65 of CM2 was mutated to alanine, and examined viral growth and viral protein synthesis in the recombinant-infected cells. The rCM2-C65A virus grew as efficiently as did the parental virus in cultured HMV-II cells as well as in embryonated chicken eggs. The synthesis and biochemical features of HEF, NP, M1 and mutant CM2 in the rCM2-C65A-infected HMV-II cells were similar to those in the parental virus-infected cells. Furthermore, membrane flotation analysis of the infected cells revealed that equal amount of viral proteins was recovered in the plasma membrane fractions of the rCM2-C65A-infected cells to that in the parental virus-infected cells. These findings indicate that defect in palmitoylation of CM2 does not affect transport and maturation of HEF, NP and M1 as well as CM2 in virus-infected cells, and palmitoylation of CM2 is dispensable to influenza C virus replication.     

Inhibition by prostaglandin PGA1 on the multiplication of influenza virus is a dose-dependent effect

Cyclopentenone prostaglandins (PGs), are strong inhibitors of the multiplicative cycle of a wide variety of enveloped RNA and DNA viruses. Their antiviral activity is generally associated with alterations in the synthesis or maturation of specific virus proteins. In this report, we describe the effect of cyclopentenone PGA1 on the replication of influenza A virus Ulster 73 in LLC-MK2 cells. PGA1 was found to inhibit viral replication in a dose-dependent fashion and virus particle yield was reduced at a PGA1 concentration, which did not suppress protein synthesis in mock-infected cells. The kinetic of late viral protein synthesis was delayed in PGA1-treated cells till 10 h post-infection; after that period, viral polypeptide synthesis appeared to be similar in PGA1-treated as well as untreated cells both infected by Ulster 73 virus. This finding suggests that PGA1 might interfere with one or more events in the viral multiplicative cycle such as protein synthesis and assembly, correct insertion of virus polypeptides into the cell membrane and, or maturation of Ulster 73 virion particles. In particular, inhibition of viral replication in LLC-MK2 cells by PGA1 is accompanied by the induction of a cellular polypeptide of 70K molecular weight. We identified this cell protein as a heat shock protein (HSP) related to the inducible isoform of HSP 70, a polypeptide of 72K molecular weight. Induction of this polypeptide by PGA1 was found to be dose-dependent and a substantial accumulation could be seen at a PGA1 concentration that did not inhibit cell protein synthesis in uninfected cells. HSP 70 synthesis started after the beginning of PGA1 treatment and remained at the same level for at least 10 h, leading us to hypothesize that the delay of production of late Ulster 73 proteins could be the consequence of HSP 70 synthesis. These results suggest that HSP 70 could play a role in the antiviral activity of cyclopentenone PGA1 in LLC-MK2 cells.     

Characterization of the human cytomegalovirus UL97 gene product as a virion-associated protein kinase

Summary. The cellular localization and virion association of the human cytomegalovirus (HCMV) UL97 protein were studied. UL97 protein demonstrated early nuclear localization followed by late perinuclear accumulation. It was found to be a structural virion constituent detected in all three enveloped forms of extracellular viral particles and shown to be phosphorylated by the virion-associated protein kinase. UL97 protein immunoprecipitated from virions and from infected cells demonstrated protein kinase activity manifested by autophosphorylation. This activity was reduced in the presence of a ganciclovir-resistance mutation at residue 460, implicated in nucleotide binding. A mutant virus, from which the proposed UL97 kinase catalytic domain had been deleted, could not be propagated in the absence of a helper wild-type virus. The characterization of UL97 protein as a virion-associated protein kinase which appears essential for viral replication, provides further insight into HCMV replication and could identify a potential novel target for antiviral therapy. Received September 2, 1997 Accepted January 14, 1998     

The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein.

To study the role of the precursor to the membrane protein (prM) in flavivirus maturation, we inhibited the proteolytic processing of the Murray Valley encephalitis (MVE) virus prM to membrane protein in infected cells by adding the acidotropic agent ammonium chloride late in the virus replication cycle. Viruses purified from supernatants of ammonium chloride-treated cells contained prM protein and were unable to fuse C6/36 mosquito cells from without. When ammonium chloride was removed from the cells, both the processing of prM and the fusion activity of the purified viruses were partially restored. By using monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of MVE virus, we found that at least three epitopes were less accessible to their corresponding antibodies in the prM-containing MVE virus particles. Amino-terminal sequencing of proteolytic fragments of the E protein which were reactive with sequence-specific peptide antisera or MAb enabled us to estimate the site of the E protein interacting with the prM to be within amino acids 200 to 327. Since prM-containing viruses were up to 400-fold more resistant to a low pH environment, we conclude that the E-prM interaction might be necessary to protect the E protein from irreversible conformational changes caused by maturation into the acidic vesicles of the exocytic pathway.     

Autographa californica multiple nucleopolyhedrovirus odv-e66 is an essential gene required for oral infectivity

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e66 is a core gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E66. The N-terminal 23 amino acid of the envelope protein ODV-E66 are sufficient to direct native and fusion proteins to induced membrane microvesicles and the viral envelope during infection with AcMNPV. In this study, an odv-e66-knockout bacmid can not express N-terminal hydrophobic domains was constructed via homologous recombination in Escherichia coli. The odv-e66 deletion had no effect on budded virus (BV) production and viral DNA replication in infected Sf9 cells. Larval bioassays demonstrated that injection of odv-e66 deletion BV into the hemocoel could kill P. xylostella larvae as efficiently as repaired and control viruses; however, odv-e66 deletion mutant resulted in a 50% lethal dose that was 10(3) higher than that of the repaired and control viruses when inoculated per os. These results indicated that ODV-E66 envelope protein most likely played an important role in the oral infectivity of AcMNPV, but is not essential for virus replication.     

Differential inhibition of cellular and Sindbis virus translation by brefeldin A

Brefeldin A is a macrolide compound that interferes with the secretory pathway and also affects protein synthesis in mammalian cells. As a result, this antibiotic impedes the maturation of viral glycoproteins of enveloped viruses and viral genome replication in several virus species. In the present work, we show that translation of subgenomic mRNA from Sindbis virus, which in contrast to cellular translation is resistant to brefeldin A after prolonged treatment. The phosphorylation of eIF2alpha as a result of brefeldin A treatment correlates with the inhibition of cellular translation, while late viral protein synthesis is resistant to this phosphorylation. The effect of brefeldin A on Sindbis virus replication was also examined using a Sindbis virus replicon. Although brefeldin A delayed viral RNA synthesis, translation by non-replicative viral RNAs was not affected, reinforcing the idea that brefeldin A delays viral RNA replication, but does not directly affect Sindbis virus protein synthesis.     

Ultrastructural study of Mayaro virus replication in BHK-21 cells

Summary The replication of Mayaro virus in BHK-21 cells was studied by electron microscopy. The infected cells show an intense vacuolization and proliferation of membranous structures. At 5 h post-infection, precursor virus particles were seen in the cytoplasm of infected cells. Later, mature virus particles were found outside the cells and budding from the plasma membrane. Enveloped virus particles were also observed inside the vesicles and budding across their membrane. The release of virus particles into the extracellular space by exocytosis was also observed. In a later stage of the infection, inclusion bodies were sometimes present in the cytoplasm of infected cells. We conclude that in BHK-21 cells, budding from the plasma membrane is the main process of Mayaro virus maturation, and in this kind of cell replication differs significantly from that observed inAedes albopictus cells.     

Unique properties of the inner core of bacteriophage phi8, a virus with a segmented dsRNA genome

The inner core of bacteriophage phi8 is capable of packaging and replicating the plus strands of the RNA genomic segments of the virus in vitro. The particles composed of proteins P1, P2, P4, and P7 can be assembled in cells of E. coli that carry plasmids with cDNA copies of genomic segment L. The gene arrangement on segment L was found to differ from that of other cystoviruses in that the gene for the ortholog of protein P7 is located at the 3' end of the plus strand rather than near the 5' end. In place of the normal location of gene 7 is gene H, whose product is necessary for normal phage development, but not necessary for in vitro genomic packaging and replication. Genomic packaging is dependent upon the activity of an NTPase motor protein, P4. P4 was purified from cell extracts and was found to form hexamers with little NTPase activity until associated with inner core particles. Labeling studies of in vitro packaging of phi8 RNA do not show serial dependence; however, studies involving in vitro packaging for the formation of live virus indicate that packaging is stringent. Studies with the acquisition of chimeric segments in live virus indicate that phi8 does package RNA in the order s/m/l. The inner core of bacteriophage phi8 differs from that of its relatives in the Cystoviridae in that the major structural protein P1 is able to interact with the host cell membrane to effect penetration of the inner core into the cell.     

Assembly of the HIV-1 core particle

The core particle of HIV-1 assembles at the membrane of the host cell as the virus buds from the surface. The structural proteins and enzymes that comprise the core are translated as part of two polyprotein precursors, Gag and GagPol. The Gag precursor contains the structural proteins of the core and is both necessary and sufficient for directing particle assembly and budding. Over the past few years, significant progress has been made in our understanding of the interactions that drive particle assembly. Specifically, determinants within the Gag precursor that direct membrane association, Gag-Gag interactions and particle budding have been identified and partially characterized. Subdomains of the host cell membrane that favor particle assembly and budding have also been described. Finally, a potential role for cellular processes in mediating the final stages in particle release has recently been proposed and a cellular protein that appears to bind directly to the Gag precursor has been identified. Each of these observations helps to clarify previously obscure aspects of viral replication and points towards potential targets for the design of novel therapies.     

Suppression of hepatitis B virus replication by SRPK1 and SRPK2 via a pathway independent of the phosphorylation of the viral core protein

The SR-domain protein kinase (SRPK) 1 and 2 are two important kinases involved in cellular RNA splicing. Recently, it was suggested that these two kinases, which could bind to the hepatitis B virus (HBV) core protein, might be the major cellular kinases that phosphorylate the core protein to regulate HBV replication. In this report, we tested the role of SRPK1 and SRPK2 in HBV replication and found that both of them could suppress HBV replication by reducing the packaging efficiency of the pgRNA without affecting the formation of the viral core particles. This suppressive effect of SRPK1 and SRPK2 on HBV replication cannot be explained by their phosphorylation activities on the HBV core protein as the over-expression of these two kinases had no detectable effects on HBV core protein phosphorylation in vivo and their mutants that lacked the kinase activity could still suppress HBV DNA replication. Thus, these findings demonstrate a negative role of SRPK1 and SRPK2 in the regulation of HBV replication through a mechanism not involving the phosphorylation of the core protein.     

Structural maturation of rubella virus in the Golgi complex

Rubella virus is a small enveloped virus that assembles in association with Golgi membranes. Freeze-substitution electron microscopy of rubella virus-infected cells revealed a previously unrecognized virion polymorphism inside the Golgi stacks: homogeneously dense particles without a defined core coexisting with less dense, mature virions that contained assembled cores. The homogeneous particles appear to be a precursor form during the virion morphogenesis process as the forms with mature morphology were the only ones detected inside secretory vesicles and on the exterior of cells. In mature virions potential remnants of C protein membrane insertion were visualized as dense strips connecting the envelope with the internal core. In infected cells Golgi stacks were frequently seen close to cytopathic vacuoles, structures identified as the sites for viral RNA replication, along with the rough endoplasmic reticulum and mitochondria. These associations could facilitate the transfer of viral genomes from the cytopathic vacuoles to the areas of rubella assembly in Golgi membranes.     

The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence

Influenza A virus particles assemble and bud from plasma membrane domains enriched with the viral glycoproteins but only a small fraction of the total M2 protein is incorporated into virus particles when compared to the other viral glycoproteins. A membrane proximal cholesterol recognition/interaction amino acid consensus (CRAC) motif was previously identified in M2 and suggested to play a role in protein function. We investigated the importance of the CRAC motif on virus replication by generating recombinant proteins and viruses containing amino acid substitutions in this motif. Alteration or completion of the M2 CRAC motif in two different virus strains caused no changes in virus replication in vitro. Viruses lacking an M2 CRAC motif had decreased morbidity and mortality in the mouse model of infection, suggesting that this motif is a virulence determinant which may facilitate virus replication in vivo but is not required for basic virus replication in tissue culture.     

pAMT11, a novel plasmid isolated from a Thermococcus sp. strain closely related to the virus-like integrated element TKV1 of the Thermococcus kodakaraensis genome

A novel extrachromosomal element that we called pAMT11 was discovered in a deep-sea vent isolate belonging to the hyperthermophilic euryarchaeal order Thermococcales. It consists of a double-stranded DNA of 20,534 bp which encodes 30 putative open reading frames (ORFs) of which six could be assigned to a putative function on the basis of sequence similarity to known genes or to protein domain families. Most of the ORFs of pAMT1 showed homology and synteny with a genomic island of Thermococcus kodakaraensis KOD1. This region, named TKV1, was previously described as a “virus-like integrated element” and assumed to integrate into the host chromosome by a site-specific recombination mechanism similar to that of Sulfolobus solfataricus virus 1. While most of the genes shared by pAMT11 and TKV1 encode putative membrane proteins presumably involved in virus particle formation, attempts to induce production of virus particles by mitomycin treatment of AMT11 cultures failed, suggesting that pAMT11 may represent the genome of a defective virus or a plasmid. Genomes of mobile elements usually contain two regions: a core of conserved genes mainly involved in replication, maintenance or spreading of the genetic element, and a variable set of accessory genes. Surprisingly, genes presumably implied in the replication process are quite divergent between TKV1 and pAMT11. Indeed, TKV1 possesses a MCM-like protein that may function as a replication initiator, while pAMT11 encodes a putative non-conventional protein distantly related to the Rep protein previously described in a small plasmid of Pyrococcus sp. strain JT1, assumed to replicate by a rolling-circle (RC) mechanism. However, in the case of pAMT11, this mode of plasmid replication could not be experimentally proven and is questionable given the lack of significant similarities with any other members of the RC-Rep superfamily and its unusual large size compared to other RC plasmids.     

Varicella-zoster virus (VZV) ORF65 virion protein is dispensable for replication in cell culture and is phosphorylated by casein kinase II, but not by the VZV protein kinases

The unique short region of varicella zoster virus (VZV) encodes four genes. One of these, ORF65, is predicted to encode an 11-kDa protein. Antibody to ORF65 protein immunoprecipitated a 16-kDa protein from the membrane fraction of VZV-infected cells. ORF65 protein was shown to be phosphorylated by casein kinase II. The VZV ORF47 or ORF66 protein kinases were not required for phosphorylation of ORF65. VZV with a large deletion in ORF65 was constructed and was shown to be dispensable for replication of virus in cell culture. The herpes simplex virus homolog of VZV ORF65 has been reported to be located in the nucleus of infected cells and in virions as a tegument protein, whereas the pseudorabies virus homolog is located in the Golgi apparatus of infected cells and in virions as a type II membrane protein. The ORF65 protein localized to the Golgi apparatus in virus-infected cells and was located in virions, most likely as a type II membrane protein. Thus, VZV ORF65 more closely resembles its pseudorabies virus homolog in its localization in infected cells and virions.