Storage of cellular 5' mRNA caps in P bodies for viral cap-snatching.

The minus strand and ambisense segmented RNA viruses include multiple important human pathogens and are divided into three families, the Orthomyxoviridae, the Bunyaviridae, and the Arenaviridae. These viruses all initiate viral transcription through the process of "cap-snatching," which involves the acquisition of capped 5' oligonucleotides from cellular mRNA. Hantaviruses are emerging pathogenic viruses of the Bunyaviridae family that replicate in the cytoplasm of infected cells. Cellular mRNAs can be actively translated in polysomes or physically sequestered in cytoplasmic processing bodies (P bodies) where they are degraded or stored for subsequent translation. Here we show that the hantavirus nucleocapsid protein binds with high affinity to the 5' cap of cellular mRNAs, protecting the 5' cap from degradation. We also show that the hantavirus nucleocapsid protein accumulates in P bodies, where it sequesters protected 5' caps. P bodies then serve as a pool of primers during the initiation of viral mRNA synthesis by the viral polymerase. We propose that minus strand segmented viruses replicating in the cytoplasm have co-opted the normal degradation machinery of P bodies for storage of cellular caps. Our data also indicate that modification of the cap-snatching model is warranted to include a role for the nucleocapsid protein in cap acquisition and storage.

HIV-1 infection despite immediate combination antiviral therapy after infusion of contaminated white cells.

We present a sixth human case in which primary human immunodeficiency virus (HIV-1) infection occurred, despite antiretroviral prophylaxis, after accidental inoculation of infected blood. In the prior five instances, variables such as large virus dose, late administration of antivirals, viral resistance to zidovudine, and pre-existent immunosuppression, may have played a role in the treatment failure. In this case, high-dosage oral zidovudine was given within minutes of the accident and replaced 2 1/2 days later with interferon alpha and dideoxyinosine (ddl). Despite aggressive treatment, HIV-1 infection was demonstrated in blood, spleen, and brain tissue at autopsy 16 days later. Of the tissues studied, detection of HIV-1 was most prominent in the spleen. Double-label immunocytochemistry confirmed the morphologic impression that while some of the infected spleen cells were CD3-positive T cells, the majority were macrophages. Thus, current single or dual (zidovudine, ddl-interferon) therapies for accidental HIV-1 inoculation may not be effective in preventing early infection. Further trials in animals appear warranted to evaluate protection by other strategies, such as passive immunity or combinations of agents that penetrate the brain and attack HIV-1 viral replication at differing sites.

Early viral brain invasion in iatrogenic human immunodeficiency virus infection.

We report a 68-year-old man who received an IV inoculation of WBCs for an indium radionuclide scan containing 600 to 700 tissue culture infectious doses of human immunodeficiency virus type 1 (HIV-1) from an HIV-1-infected individual. The recipient immediately received zidovudine, then was switched to dideoxyinosine and interferon-alpha, but died of hepatorenal syndrome and hepatic encephalopathy 15 days later. HIV-1 cultures were positive from the recipient's blood on day 14 but not days 0, 1, and 8. At autopsy, cultures of parietal lobe isolated HIV-1. HIV-1 nucleic acid was present in several brain areas, but not in several other organs, by two independent laboratories using the polymerase chain reaction. The brain showed mild perivascular cuffing and a mild lymphocytic meningitis, but there was no evidence of glial nodules, giant cells, or white matter abnormalities. HIV-1 pg41 viral antigen was seen by immunoperoxidase staining in rare infiltrating cells within perivascular and subpial spaces. Thus, HIV-1 was isolated from brain 15 days after mistaken HIV-1 inoculation and 1 day after virus was first recovered from blood.

Cosegregation of the renin allele of the spontaneously hypertensive rat with an increase in blood pressure.

The spontaneously hypertensive rat (SHR) exhibits alterations in the renin-angiotensin-aldosterone system which are similar to those that characterize patients with "nonmodulating" hypertension, a common and highly heritable form of essential hypertension. Accordingly, we determined whether the inheritance of a DNA restriction fragment length polymorphism (RFLP) marking the renin gene of the SHR was associated with greater blood pressure than inheritance of a RFLP marking the renin gene of a normotensive control rat. In an F2 population derived from inbred SHR and inbred normotensive Lewis rats, we found the blood pressure in rats that inherited a single SHR renin allele to be significantly greater than that in rats that inherited only the Lewis renin allele. To the extent that the SHR provides a suitable model of "nonmodulating" hypertension, these findings raise the possibility that a structural alteration in the renin gene, or a closely linked gene, may be a pathogenetic determinant of increased blood pressure in one of the most common forms of essential hypertension in humans.

Genetic analysis of familial isolated growth hormone deficiency type I.

Nuclear DNA from individuals belonging to nine different families in which two sibs were affected with isolated growth hormone deficiency type I were studied by restriction endonuclease analysis. By using 32P-labeled human growth hormone or the homologous human chorionic somatomammotropin complementary DNA (cDNA) sequences as a probe, the growth hormone genes of affected individuals from all families yielded normal restriction patterns. Polymorphic restriction endonuclease sites (HincII and MspI), which are closely linked to the structural gene for growth hormone on chromosome 17, were used as markers in linkage analysis of DNA of family members. Of the nine affected sib pairs two were concordant, three were possibly concordant, and four were discordant for both linked markers. Since only concordant sib pairs would have inherited the same growth hormone alleles, further studies to identify mutations of the growth hormone genes should be limited to this subgroup. It is unlikely that the discordance observed in four of the sib pairs is due to recombination, because the polymorphic HincII site is only 116 base-pairs from the -26 codon of the growth hormone gene. Thus, in at least four of the nine families, the mutation responsible for isolated growth hormone deficiency is not within or near the structural gene for growth hormone on chromosome 17.

Relative levels of methylation in human growth hormone and chorionic somatomammotropin genes in expressing and non-expressing tissues.

It has been shown that the extent of methylation of cytosine in vertebrate DNA is inversely correlated with gene expression. We studied cytosine methylation in and around the homologous human growth hormone (GH) and chorionic somatomammotropin (CS) genes to determine if these genes are undermethylated in DNA from tissues in which they are expressed (pituitary and placenta, respectively) compared to other tissues. Hpa II and Hha I (which cleave only unmethylated 5' CCGG 3' and 5' GCGC 3' respectively) and Msp I (which cleaves CCGG and CmeCGG) were used to digest DNA samples followed by gel electrophoresis, Southern transfer and hybridization with a GH cDNA probe. The extent of methylation of Hpa II and Hha I sites in the GH and CS genes was leukocyte much greater than pituitary greater than placenta = hydatidiform mole. Taken as a whole, our data support the hypothesis that undermethylation is a necessary but not sufficient condition for gene expression since placental and pituitary DNAs are less methylated than leukocyte DNA in this region. However, the correlation between gene expression and undermethylation is imperfect since (1) hydatiform mole DNA has a very similar methylation pattern compared to placental DNA even though moles make little or no CS and (2) the level of methylation of the GH gene compared to the CS gene does not vary in a tissue-specific manner.

Homozygous alpha thalassemia/Hb G Philadelphia.

Microcytic red cells from a 70 year old Negro man with mild anemia contained only hemoglobin G-Philadelphia. Red cells from all of his children had low-normal MCV's, and contained 32-34 percent of the abnormal hemoglobin. Oxygen affinity of his blood and stability of his hemolysate were normal, suggesting that his mild anemia was not caused by the the abnormal hemoglobin. Restriction endonuclease analyses of DNA from the proband and his offspring showed that the alpha G-Philadelphia globin gene exists in only one copy per chromosome. The new gene was probably created by an unequal cross-over which deleted an alpha globin coding sequence (derived from one or both alpha globin genes), as well as some or all of the DNA sequence between those genes.

Molecular basis for familial isolated growth hormone deficiency.

Nuclear DNA from four individuals with familial isolated growth hormone (somatotropin) deficiency (IGHD) type A was studied by restriction endonuclease analysis. By using 32P-labeled human growth hormone (hGH) cDNA sequences as a probe, patterns seen after various digestions indicated that these individuals were homozygous for a deletion of at least 7.5 kilobases (kb) of DNA. This deletion includes the gene that encodes the normal growth hormone but does not include the variant growth hormone gene. Restriction patterns of DNAs from all family members agreed with an autosomal recessive mode of inheritance of the deletion that correlates with the clinical phenotype. Furthermore, independent assortment of the two types of hGH genes suggests that these genes are nonallelic. These findings indicate that, in these families, IGHD type A is caused by deletion of the normal hGH genes and that this disorder can occur in the presence of variant hGH genes.