The human skeletal muscle adenine nucleotide translocator gene maps to chromosome 4q35 in the region of the facioscapulohumeral muscular dystrophy locus

The large number of sequenced clones of HIV-1 and related viruses made it possible to indicate conserved elements with potential regulatory or structural functions. Such analysis was combined with directed mutagenesis in order to investigate the importance of elements that may influence the initiation of plus-strand DNA synthesis. The main site for plus-strand initiation is a polypurine tract near the 3' end of the viral RNA (the 3' PPT). An exact copy of this PPT is located in the middle of the genome (the internal PPT). Upstream from the internal PPT there is an inverted repeat. Mutants designed to upset the internal PPT (i.e., purine to pyrimidine changes), as well as mutants designed to abolish the potential stem-loop formation (changes around the internal PPT or in the upstream inverted repeat) both resulted in viruses with a reduced ability to replicate. Upsetting the stem-loop formation was, however, less harmful than changing the polypurine nature of the PPT. Changing a conserved T on the 3' side of the PPT to a C did not affect the phenotype.     

3' Terminal putative stem-loop structure required for the accumulation of cymbidium ringspot viral RNA

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus (CymRSV) was used to identify the cis-acting nature of the last 77-nt sequence of the viral genome which is required for DI RNA accumulation. The 3'-terminal cis-essential domain of both genomic and DI RNAs can be folded into a stable stem-loop structure composed of three hairpins and two short non-base-paired regions. None of the three conserved stem-loops can be deleted without abolishing the infectivity of DI RNA. Similarly, those mutants in which base-paired stem regions were disrupted by single-, double-, or triple-base substitutions were unable to replicate. However, when the original structures were reconstructed by compensatory mutations the viability of the molecules was also restored. Limited mutation (1 or 2 nt) in the non-base-paired region did not show any significant effect on viral replication. Our results strongly suggest that the proposed structure for the 3' terminus of the viral genome is very important for viral RNA replication. It is very likely that the function of this structure is to promote the minus-strand synthesis of CymRSV DI RNA. Evidence is provided that the proposed 3'-terminal structure is relevant not only for CymRSV DI but for genomic RNA as well.     

Sequence requirements of the Epstein-Barr virus latent origin of DNA replication

The Epstein-Barr virus (EBV) latent origin of DNA replication (oriP) is composed of two elements that contain binding sites for the sole viral gene product required for latent cycle replication, EBNA-1. One of these elements, region I, functions as an EBNA-1-dependent enhancer for RNA polymerase II-transcribed genes, may play a role in plasmid segregation, and is required for origin function in B cells latently infected with EBV. The second element, region II, contains or is very near the site of initiation of DNA replication. A genetic approach was taken to determine the contribution of the EBNA-1 binding sites in oriP to origin function. Although region I is required for the transient replication of plasmids bearing region II in EBV-infected B cells, a plasmid lacking region I but containing region II, was observed to replicate transiently in both D98/Raji and HeLa cells expressing EBNA-1. Thus, binding of EBNA-1 to region I is not absolutely required for the molecular events that lead to initiation of DNA replication at region II. Site-directed mutagenesis of the four EBNA-1-binding sites in region II, individually and in various combinations, demonstrated that only two EBNA-1-binding sites are required for region II function. The results obtained with these mutants, together with the analysis of the replicative ability of plasmids containing insertions between EBNA-1-binding sites, suggested that the spatial relationship of the two sites is critical. Mutants that contain only two EBNA-1-binding sites separated by 26 to 31 bp in region II were not maintained as plasmids over many cell generations and were greatly reduced in their ability to replicate transiently in D98/Raji cells. The EBNA-1-induced bending or untwisting of the DNA in EBNA-1-binding sites 1 and 4 in region II did not, however, demonstrate this spatial constraint. It may be concluded from these results that specific protein-protein interactions between EBNA-1 and/or between EBNA-1 and a cellular protein(s) are required for origin function.     

Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene

The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.     

Evaluation of pedicle screw insertion monitored by intraoperative evoked electromyography

The clinical importance of hepatitis B virus (HBV) genome variability has been reported recently. One example is the occurrence of hepatitis B virus pre-core mutants, which arise during spontaneous or interferon-induced seroconversion from HBeAg to anti-HBe and are thought to be selected by immune pressure. A survey of HBV pre-core mutants and viral genotypes in 35 HBeAg negative patients during interferon therapy was carried out to understand viral pathogenesis in this form of chronic hepatitis B. Seventeen patients responded to interferon therapy as assessed by the sustained normalization of serum ALT levels and the significant decrease of viremia levels. The response rate to interferon was independent of both initial serum viral DNA level and interferon doses. During interferon therapy, a significant decrease of M0 (wild-type pre-core sequence at pos. 1887-1908), M1 (TGG to TAG at pos. 1896) or M2 (TGG to TAG at pos. 1896, and GGC to GAC at pos. 1899) positive viral genomes was found in 48%, 42%, and 33% of patients, respectively. A higher response rate to interferon therapy was observed in patients infected with HBV genotype A (70%) or M0 positive strains (75%) as compared to patients infected with genotype D/E (40%) or M1/M2 positive strains (44%). The data support the hypothesis that pre-core defective HBV represent viral mutants with an increased capacity to resist exogenous alpha interferon. These findings emphasize that characterization of HBV genome variability prior to interferon therapy may help to predict antiviral response in HBeAg negative patients.     

Insulation lead failure: is it a matter of insulation coating, venous approach, or both?

Gag gene mutants of human immunodeficiency virus type 1 (HIV-1) were analyzed for their potentials of inhibiting the replication of wild-type (wt) HIV-2, the second AIDS virus, in a single-round of viral replication. Of twenty-two HIV-1 gag mutants examined, seven were found to efficiently interfere with the replication of wt HIV-2. Some mutants, which can suppress the replication of wt HIV-1, did not show this inhibitory effect. These mutants were defective at the late phase of viral replication. A mutant designated NL-C1a was demonstrated to be very effective against the replication of HIV-1 and HIV-2 in monocytic cells as well as in lymphocytic cells.     

The detection and characterization of multiple hemagglutinin-esterase (HE)-defective viruses in the mouse brain during subacute demyelination induced by mouse hepatitis virus

It has previously been shown that passive immunization with antibodies specific for the hemagglutinin-esterase (HE) protein of mouse hepatitis virus (MHV) prevents acute lethal encephalitis, resulting in a subacute and chronic demyelination in mice infected with JHM(2), an isolate of the neurotropic JHM strain of MHV. To determine possible genetic changes occurring during infection, viruses were isolated from the brain of infected mice at various time points after infection and examined for the patterns of their structural proteins. The results showed that the sizes and expression levels of the viral spike, membrane, and nucleocapsid proteins were constant among 161 virus isolates throughout the infection. In contrast, most of the viruses isolated later in infection did not synthesize HE protein. This finding suggests that the HE gene expression is extremely variable and is preferentially lost during prolonged viral infections. In contrast, when viruses were passaged in tissue culture, no significant accumulation of HE protein-defective mutants was observed, suggesting that the accumulation of HE protein-defective mutants in infected animals was most likely the result of the positive selection for these mutants during the subacute and chronic infection. The genetic defects of HE gene in these mutants were characterized by cloning, sequencing, and in vitro translation of HE genes. Most of the mutations in the HE protein-defective mutants consisted of deletions of various lengths at different sites within the HE-coding region, resulting in the change of the reading frame and early termination. However, most of the truncated HE proteins were not detected in the infected cells. Since viruses from different mice exhibited different types of defects, the HE mutations probably occurred de novo in the brain. These results demonstrated the exceptionally rapid selection of HE-defective mutants during viral infection of mice and suggest that their selection may be related to the neuropathogenicity or persistence of MHV.     

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.     

p53 status does not determine outcome of E1B 55-kilodalton mutant adenovirus lytic infection

The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G1. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39 degreesC, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39 degreesC. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.