Viral pro-survival proteins block separate stages in Bax activation but changes in mitochondrial ultrastructure still occur.

Mitochondrial dysfunction mediated by Bax and Bak is a critical step in mammalian cell apoptosis. However, the molecular mechanism of Bax activation remains unknown and has been difficult to investigate due to its rapid and stochastic nature. It is currently unclear whether mitochondria play a passive role in the initiation of apoptosis, remaining unaffected by cell stresses until Bax and Bak are active, or whether they actively participate in Bax/Bak activation. Here, two viral proteins, E1B19K and BHRF1, are examined for their ability to block Bax activation at different steps and thereby reveal the timing of mitochondrial changes during apoptosis. We demonstrate that BHRF1 strongly inhibits Bax activation but not upstream apoptotic signaling events, while E1B19K permits initial stages of Bax activation but prevents the subsequent oligomerization of Bax that is required for mitochondrial dysfunction. In this defined system we show that changes in mitochondrial ultrastructure, characteristic of cells undergoing apoptosis, precede Bax activation and are not blocked by E1B19K and BHRF1. We suggest that the ability of mitochondria to respond to apoptotic stress prior to Bax activation indicates that these organelles may play a direct role in activating Bax.

Efficient initiation of HCV RNA replication in cell culture.

Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-alpha rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.

Mmf1p, a novel yeast mitochondrial protein conserved throughout evolution and involved in maintenance of the mitochondrial genome.

A novel protein family (p14.5, or YERO57c/YJGFc) highly conserved throughout evolution has recently been identified. The biological role of these proteins is not yet well characterized. Two members of the p14.5 family are present in the yeast Saccharomyces cerevisiae. In this study, we have characterized some of the biological functions of the two yeast proteins. Mmf1p is a mitochondrial matrix factor, and homologous Mmf1p factor (Hmf1p) copurifies with the soluble cytoplasmic fraction. Deltammf1 cells lose mitochondrial DNA (mtDNA) and have a decreased growth rate, while Deltahmf1 cells do not display any visible phenotype. Furthermore, we demonstrate by genetic analysis that Mmf1p does not play a direct role in replication and segregation of the mtDNA. rho(+) Deltammf1 haploid cells can be obtained when tetrads are directly dissected on medium containing a nonfermentable carbon source. Our data also indicate that Mmf1p and Hmf1p have similar biological functions in different subcellular compartments. Hmf1p, when fused with the Mmf1p leader peptide, is transported into mitochondria and is able to functionally replace Mmf1p. Moreover, we show that homologous mammalian proteins are functionally related to Mmf1p. Human p14.5 localizes in yeast mitochondria and rescues the Deltammf1-associated phenotypes. In addition, fractionation of rat liver mitochondria showed that rat p14.5, like Mmf1p, is a soluble protein of the matrix. Our study identifies a biological function for Mmf1p and furthermore indicates that this function is conserved between members of the p14.5 family.

Salvage after severe lower-extremity trauma: are the outcomes worth the means?

Advances in reconstructive surgery have allowed for impressive salvage after severe lower-extremity trauma but not without complications when compared with immediate below-knee amputation. Several amputation index scores have been developed to help predict successful salvage as defined by a viable rather than a functional extremity. The purpose of this study was to evaluate retrospectively the predictive value of the amputation index scores and to assess prospectively overall health status and specific dysfunction in successful limb salvage and primary and secondary amputation by administering standardized generic and specific outcomes questionnaires (Medical Outcomes Study 36-Item Short-Form Health Survey, Western Ontario and MacMaster Universities Osteoarthritis Index). A retrospective chart review identified 55 severe lower-extremity injuries (Gustilo Type IIIB and IIIC) over a 12-year period (1984 to 1996). Forty-six severe open tibial fractures in 45 patients underwent attempted salvage. All required soft-tissue coverage by either local or free flap or vascular repair for leg salvage. The attempted-salvage group was subdivided into successful salvage and secondary amputation. The other nine patients underwent a primary amputation. There were no statistically significant differences in terms of patient demographics or other injuries (Injury Severity Score) in the three groups. Forty-eight of 54 patients with an average 5-year follow-up completed a validated generic and specific outcomes health questionnaire. In the attempted-salvage group, 89 percent of patients had a successful salvage and 11 percent came to a secondary amputation. The amputation index scores correctly predicted an amputation in 32 percent of patients. The magnitude of the amputation index scores did not correlate with the physical outcomes scores and were not found to add any significant value of information to the surgeon's decision making. Patients undergoing primary and secondary amputation had a worse physical outcomes score (28 versus 38) than successful salvage (p < 0.007). Even so, the SF-36 (physical component score) outcomes score for this group of injured extremities, regardless as to whether salvaged or amputated, was as low as or lower than that of many serious medical illnesses, suggesting that severe lower-extremity trauma impairs health as much as or more than being seriously ill. The mental component score in this group was comparable to that of a healthy population (49 versus 50), which implies the disability is primarily physical rather than psychological. Ninety-two percent of patients preferred their salvaged leg to an amputation at any stage of their injury, and none would have preferred a primary amputation.

Molecular virology of hepatitis C virus: an update with respect to potential antiviral targets.

Hepatitis C virus (HCV), a positive-strand enveloped RNA virus, is a major cause of chronic liver disease worldwide. Cis-acting RNA elements and virus-encoded polypeptides required for HCV replication represent attractive targets for the development of antiviral therapies. Internal ribosome entry site-directed translation of HCV genome RNA produces a long polyprotein which is co- and post-translationally processed to yield at least 10 viral proteins. A host signal peptidase is responsible for maturation of the structural proteins located in the N-terminal one-third of the polyprotein. Thus far, four enzymatic activities encoded by the non-structural (NS) proteins have been reported. The NS2-3 region encodes an autoproteinase responsible for cleavage at the 2/3 site. The N-terminal one-third of NS3 functions as the catalytic subunit of a serine proteinase which cleaves at the 3/4A, 4A/4B, 4B/5A and 5A/5B sites, and NS4A is an essential cofactor for some of these cleavages. NS3 also encodes an RNA-stimulated NTPase/RNA helicase at its C terminus, and NS5B has been shown to possess an RNA-dependent RNA polymerase activity. To date, no functions have been reported for NS4B or NS5A in RNA replication, however, NS5A has been implicated in modulating the sensitivity of HCV to interferon. Sequence and structural conservation within the 3' terminal 98 bases of genomic RNA suggest a functional importance in the virus life-cycle and hence another target for antiviral intervention. Recently, HCV infection was shown to be initiated in chimpanzees following intrahepatic inoculation of RNA transcribed from cloned HCV cDNA. The ability to generate large quantities of infectious HCV RNA may facilitate the development of reliable cell culture replication systems useful for the evaluation of antiviral drugs.

Secondary structure determination of the conserved 98-base sequence at the 3' terminus of hepatitis C virus genome RNA.

The RNA genome of hepatitis C virus (HCV) terminates with a highly conserved 98-base sequence. Enzymatic and chemical approaches were used to define the secondary structure of this 3'-terminal element in RNA transcribed in vitro from cloned cDNA. Both approaches yielded data consistent with a stable stem-loop structure within the 3'-terminal 46 bases. In contrast, the 5' 52 nucleotides of this 98-base element appear to be less ordered and may exist in multiple conformations. Under the experimental conditions tested, interaction between the 3' 98 bases and upstream HCV sequences was not detected. These data provide valuable information for future experiments aimed at identifying host and/or viral proteins which interact with this highly conserved RNA element.

Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.

More than 1% of the world's population is chronically infected with hepatitis C virus (HCV). HCV infection can result in acute hepatitis, chronic hepatitis, and cirrhosis, which is strongly associated with development of hepatocellular carcinoma. Genetic studies of HCV replication have been hampered by lack of a bona fide infectious molecular clone. Full-length functional clones of HCV complementary DNA were constructed. RNA transcripts from the clones were found to be infectious and to cause disease in chimpanzees after direct intrahepatic inoculation. This work defines the structure of a functional HCV genome RNA and proves that HCV alone is sufficient to cause disease.

The human papillomavirus type 16 E7 protein is associated with the nucleolus in mammalian and yeast cells.

In this study we show, by immunofluorescence and electron microscopy immuno-gold labelling, that the major transforming protein of Human Papillomavirus type 16 E7 is associated with the nucleolus of cells derived from the HPV16-positive cervical carcinoma line CaSki. The E7 nucleolar staining appeared to be cell cycle dependent, being considerably reduced in the G2 phase. The total level of the protein in the cell, however, remained constant during all phases. We also show that the cellular protein Rb1, which is targeted by E7, is localised in the nucleus and nucleolus in CaSki cells. Thus, it is possible that the presence of E7 in the nucleolus correlates with a hypothetical function(s) of Rb1 in this particular intranuclear compartment. The nucleolar localisation of HPV16 E7 protein was also observed in the fission yeast Schizosaccharomyces pombe, suggesting that a targeting mechanism of HPV16 E7 protein into the nucleolus is common to both mammalian and yeast systems. Nucleolar localisation of HPV16 E7 protein may be independent from Rb1 since no Rb1 related proteins have been identified in fission yeast.

Formation of native hepatitis C virus glycoprotein complexes.

The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.

Recombination associated with replication of malarial mitochondrial DNA.

Mitochondrial DNA of the malarial parasite Plasmodium falciparum comprises approximately 20 copies per cell of a 6 kb genome, arranged mainly as polydisperse linear concatemers. In synchronous blood cultures, initiation of mtDNA replication coincides with the start of the 4-5 doublings in nuclear DNA that mark the reproductive phase of the erythrocytic cycle. We show that mtDNA replication coincides with a recombination process reminiscent of the replication mechanism used by certain bacteriophages and plasmids. The few circular forms of mtDNA which are also present do not replicate by a theta mechanism, but are themselves the product of recombination, and we propose they undergo rolling circle activity to generate the linear concatemers.

Absence of double-stranded replicative forms of HCV RNA in liver tissue from chronically infected patients.

The mechanism of hepatitis C virus (HCV) replication is unknown, although the classification of HCV in the Flaviviridae has led to the postulation that HCV may adopt a replication strategy similar to that of the flaviviruses. To determine if HCV double-stranded replicative forms, consistent with this strategy, were present in total liver RNA extracted from HCV-infected individuals, HCV-specific RNA was detected by reverse transcription followed by polymerase chain reaction (RT-PCR). Initially, a strand-specific RT-PCR resulting from chemical modification of the 3' end of the RNA was established using in vitro transcribed HCV RNA. This procedure allowed the specific detection of positive and negative HCV RNA strands in HCV-infected liver tissue. The species of HCV RNA was then examined in RNA extracted from liver tissue from naturally infected individuals; total liver RNA was either: (i) fractionated with 2M LiCl (designed to precipitate single-stranded and partially double-stranded RNA); or (ii) digested with RNase A in high salt conditions (designed to digest single-stranded RNA only). Amplification of positive sense HCV RNA from the LiCl-insoluble fraction, but not from the LiCl-soluble fraction nor in the RNase A-digested sample, was consistent with the interpretation that single-strand, but not double-stranded HCV RNA, was contained in the liver samples. Thus, it is unclear if a double-stranded RNA species is formed during the HCV replication cycle.

Detection and distribution of hepatitis C-specific antigens in naturally infected liver.

Hepatitis C virus antigen expression was examined using peptide antibodies in liver tissue taken at biopsy from four chronic carriers of hepatitis C virus. Hepatitis C virus antigens E2/NS1, NS3, NS4 and NS5 were widespread in unfixed frozen liver sections and were present as distinct granules or foci within the cytoplasm of hepatocytes and in infiltrating lymphocytes in portal tracts. Fixation of frozen sections with 1% formalin improved the histological appearance of the tissue section without reducing the sensitivity of antigen detection. However, in tissue sections fixed in acetone, chloroform, carbon tetrachloride or methyl carnoys, detection of all hepatocyte-specific hepatitis C virus antigens was significantly reduced. Dual immunostaining of liver sections for lymphocyte cluster of differentiation markers and hepatitis C virus antigens determined that a high proportion of cluster of differentiation 20-positive B cells and cluster of differentiation 4-and cluster of differentiation 8-positive T cells, predominant in lymphoid aggregates, were positive for hepatitis C virus antigens.

Immunohistochemical detection of the NS4 antigen of hepatitis C virus and its relation to histopathology.

The immunohistochemical localization of the hepatitis C virus (HCV) nonstructural antigen 4 (NS4) was investigated in formalin-fixed human liver biopsy samples taken from 10 patients who were anti-HCV positive. NS4 was detected within the cytoplasm of hepatocytes in all HCV-positive patients studied, but not in the mononuclear cell infiltrates, bile duct epithelium, or endothelial cells. A high proportion of hepatocytes appeared positive, but the staining intensity was variable. After a coded histological evaluation of the liver tissue, the pattern of liver injury was shown to have no significant correlation with antigen-positive hepatocytes, and no direct relationship was observed between the distribution of antigen-positive hepatocytes and areas of hepatocyte necrosis. The staining pattern was considered to be specific because liver samples from patients chronically infected with hepatitis B virus or from uninfected individuals were negative. Furthermore, no staining was noted when either preimmune rabbit serum or anti-NS4 adsorbed against the specific synthetic peptide was substituted for the primary antibody.

Localisation of hepatitis C virus proteins in infected liver tissue by immunofluorescence.

Hepatitis C virus (HCV) antigen expression was examined by immunohistochemical staining in liver tissue taken at biopsy from 8 anti-HCV positive patients. Frozen liver sections were stained by indirect immunofluorescence for capsid, E2/NS1, NS3, NS4 and NS5 using polyclonal antibodies raised to synthetic peptides from these regions. The antigens E2 and NS3 were localised in scattered hepatocytes and also in cells within and around areas of inflammation. A weaker signal was observed for NS4 and NS5 and no signal was seen for capsid antigen. No staining was seen in liver tissue from 9 individuals, including 3 hepatitis B virus-positive and 2 hepatitis delta virus/positive patients, who were negative for serological markers of HCV. The specificity of the staining reaction was also confirmed by the lack of staining in HCV-positive liver samples, after the antisera was pre-adsorbed against the specific peptide. Collectively, the data suggests that HCV may not only be hepatotropic but also lymphotropic, and this may be an important factor in the pathogenesis of HCV infection.