The role of osmiophilic bodies and Pfg377 expression in female gametocyte emergence and mosquito infectivity in the human malaria parasite plasmodium falciparum

Osmiophilic bodies are membrane-bound vesicles, found predominantly in Plasmodium female gametocytes, that become progressively more abundant as the gametocyte reaches full maturity. These vesicles lie beneath the subpellicular membrane of the gametocyte, and the release of their contents into the parasitophorous vacuole has been postulated to aid in the escape of gametocytes from the erythrocyte after ingestion by the mosquito. Currently, the only protein known to be associated with osmiophilic bodies in Plasmodium falciparum is Pfg377, a gametocyte-specific protein expressed at the onset of osmiophilic body development. Here we show by targeted gene disruption that Pfg377 plays a fundamental role in the formation of these organelles, and that female gametocytes lacking the full complement of osmiophilic bodies are significantly less efficient both in vitro and in vivo in their emergence from the erythrocytes upon induction of gametogenesis, a process whose timing is critical for fertilization with the short-lived male gamete. This reduced efficiency of emergence explains the significant defect in oocyst formation in mosquitoes fed blood meals containing Pfg377-negative gametocytes, resulting in an almost complete blockade of infection.

Alteration of the proline at position 7 of the HIV-1 spacer peptide p1 suppresses viral infectivity in a strain dependent manner

The HIV-1 spacer peptide p1 is located in the C-terminus of the Gag polyprotein and separates the nucleocapsid (NC) and p6(Gag). Research centered on p1 has been limited and as yet no function has been ascribed to this spacer peptide. We have previously found that the conserved p1 proline residues (position 7 and 13) are critical for replication in the HIV-1 strain HXB2-BH10. In this study we have focused on the proline rich p1-p6(Gag) C-terminus of HIV-1. We individually examined the role of p1 proline's in multiple strains of HIV-1 and investigated the role of three proline residues in p6(Gag) (P24, P25 and P30). Assessment of the HXB2-BH10 based mutants revealed that Gag-Pol incorporation relative to Gag decreased in the p1 mutant virions, with the double proline mutant the most impaired. Mutating both p1 proline residues was found to abolish infectivity in multiple strains of HIV-1. Independent mutation of the p1 proline at position 7 resulted in a strain-dependent suppression of viral infectivity. This defect correlates with the presence of a tyrosine residue at position 9 of p1 and occurs in the early phase of the HIV-1 replication cycle. The p1 proline residues were found to be functionally distinct from P24, P25 and P30 in p6(Gag). This work affords novel insights into our understanding of the role of p1 in HIV-1 replication.

DNA found in human immunodeficiency virus type 1 particles may not be required for infectivity

We have studied the presence and significance of retroviral genome-derived DNA in the core of human immunodeficiency virus (HIV) particles produced from transfections of HXB2 expression vectors in COS-7 cells and from HIV type 1 IIIB chronically infected H9 cells. Viruses purified by sucrose cushion centrifugation and treated with DNase I contained 1000-fold more viral RNA than DNA. However protease-defective viruses that contained only pl60 ga~p°z had less than 100 times the amount of DNA in their cores than wild-type viruses suggesting that the p66/p51 form of reverse transcriptase was responsible for DNA transcription. Viruses produced by transfections in the presence of 3'-azido-3'-deoxythymidine (AZT) contained the viral RNA genome but only DNA of premature length because of the chain terminating effects of AZT. However such viruses were as infectious for CD4 + cells as wild-type virus. We conclude that retrovirus-derived DNA in HIV-1 particles is not required for infection and does not play a significant role in this process.

Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization

A stem-loop termed the kissing-loop hairpin is one of the most highly conserved structures within the leader of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus genomic RNA. Because it plays a key role in the in vitro dimerization of short HIV-1 RNA transcripts (M. Laughrea and L. Jette, Biochemistry 35:1589-1598, 1996, and references therein; M. Laughrea and L. Jette, Biochemistry 35:9366-9374, 1996, and references therein) and because dimeric RNAs may be preferably encapsidated into the HIV-1 virus, alterations of the kissing-loop hairpin might affect the in vivo dimerization and encapsidation processes. Accordingly, substitution and deletion mutations were introduced into the kissing-loop hairpin of an infectious HIV-1 molecular clone in order to produce viruses by transfection methods. The infectivity of the resulting viruses was decreased by at least 99%, the amount of genomic RNA packaged per virus was decreased by 50 to 75%, and the proportion of dimeric genomic RNA was reduced from >80 to 40 to 50%, but the dissociation temperature of the genomic RNA was unchanged. There is evidence suggesting that the deletion mutations moderately inhibited CAp24 production but had no significant effect on RNA splicing. These results are consistent with the kissing-loop model of HIV-1 RNA dimerization. In fact, because intracellular viral RNAs are probably more concentrated in transfected cells than in cells infected by one virus and because the dimerization and encapsidation processes are concentration dependent, it is likely that much larger dimerization and encapsidation defects would have been manifested within cells infected by no more than one virus.

Maintenance of the gag/gag-pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity

Production of the human immunodeficiency virus type 1 (HIV_-1) Gag-Pol precursor protein results from a −1 ribosomal frameshifting event. In infected cells, this generates Gag and Gag-Pol in a ratio that is estimated to be 20:1, a ratio that is conserved among retroviruses. To examine the impact of this ratio on HIV_-1 replication and viral assembly, we altered the Gag/Gag-Pol ratio in virus-producing cells by cotransfecting HIV_-1 proviral DNA with an HIV_-1 Gag-Pol expression vector. Two versions of the Gag-Pol expression vector were used; one contains an active protease [PR(+)], and the other contains an inactive protease [PR(−)]. In an attempt to produce viral particles with Gag/Gag-Pol ratios ranging from 20:21 to 20:1 (wild type), 293T cells were cotransfected with various ratios of wild-type proviral DNA and proviral DNA from either Gag-Pol expression vector. Viral particles derived from cells with altered Gag/Gag-Pol ratios via overexpression of PR(−) Gag-Pol showed a ratio-dependent defect in their virion protein profiles. However, the defects in virion infectivity were independent of the nature of the Gag-Pol expression vector, i.e., PR(+) or PR(−). Based on equivalent input of reverse transcriptase activity, we estimated that HIV_-1 infectivity was reduced 250- to 1,000-fold when the Gag/Gag-Pol ratio in the virion-producing cells was altered from 20:1 to 20:21. Although virion RNA packaging was not affected by altering Gag/Gag-Pol ratios, changing the ratio from 20:1 to 20:21 progressively reduced virion RNA dimer stability. The impact of the Gag/Gag-Pol ratio on virion RNA dimerization was amplified when the Gag-Pol PR(−) expression vector was expressed in virion-producing cells. Virions produced from cells expressing Gag and Gag-Pol PR(−) in a 20:21 ratio contained mainly monomeric RNA. Our observations provide the first direct evidence that, in addition to proteolytic processing, the ratio of Gag/Gag-Pol proteins is also important for RNA dimerization and that stable RNA dimers are not required for encapsidation of genomic RNA in HIV_-1.

Proline residues within spacer peptide p1 are important for human immunodeficiency virus type 1 infectivity, protein processing, and genomic RNA dimer stability

The full-length human immunodeficiency virus type 1 (HIV-1) mRNA encodes two precursor polyproteins, Gag and GagProPol. An infrequent ribosomal frameshifting event allows these proteins to be synthesized from the same mRNA in a predetermined ratio of 20 Gag proteins for each GagProPol. The RNA frameshift signal consists of a slippery sequence and a hairpin stem-loop whose thermodynamic stability has been shown in in vitro translation systems to be critical to frameshifting efficiency. In this study we examined the frameshift region of HIV-1, investigating the effects of altering stem-loop stability in the context of the complete viral genome and assessing the role of the Gag spacer peptide p1 and the GagProPol transframe (TF) protein that are encoded in this region. By creating a series of frameshift region mutants that systematically altered the stability of the frameshift stem-loop and the protein sequences of the p1 spacer peptide and TF protein, we have demonstrated the importance of stem-loop thermodynamic stability in frameshifting efficiency and viral infectivity. Multiple changes to the amino acid sequence of p1 resulted in altered protein processing, reduced genomic RNA dimer stability, and abolished viral infectivity. The role of the two highly conserved proline residues in p1 (position 7 and 13) was also investigated. Replacement of the two proline residues by leucines resulted in mutants with altered protein processing and reduced genomic RNA dimer stability that were also noninfectious. The unique ability of proline to confer conformational constraints on a peptide suggests that the correct folding of p1 may be important for viral function.

The raft-promoting property of virion-associated cholesterol, but not the presence of virion-associated brij 98 rafts, is a determinant of human immunodeficiency virus type 1 infectivity

Lipid rafts are enriched in cholesterol and sphingomyelin and are isolated on the basis of insolubility in detergents, such as Brij 98 and Triton X-100. Recent work by Holm et al. has shown that rafts insoluble in Brig 98 can be found in human immunodeficiency virus type 1 (HIV-1) virus-like particles, although it is not known whether raft-like structures are present in authentic HIV-1 and it is unclear whether a virion-associated raft-like structure is required for HIV replication. Independently, it was previously reported that virion-associated cholesterol is critical for HIV-1 infectivity, although the specific requirement of virion cholesterol in HIV-1 was not examined. In the present study, we have demonstrated that infectious wild-type HIV-1 contains Brij 98 rafts but only minimal amounts of Triton X-100 rafts. To directly assess the functional requirement of virion-associated rafts and various features of cholesterol on HIV-1 replication, we replaced virion cholesterol with exogenous cholesterol analogues that have demonstrated either raft-promoting or -inhibiting capacity in model membranes. We observed that variable concentrations of exogenous analogues are required to replace a defined amount of virion-associated cholesterol, showing that structurally diverse cholesterol analogues have various affinities toward HIV-1. We found that replacement of 50% of virion cholesterol with these exogenous cholesterol analogues did not eliminate the presence of Brij 98 rafts in HIV-1. However, the infectivity levels of the lipid-modified HIV-1s directly correlate with the raft-promoting capacities of these cholesterol analogues. Our data provide the first direct assessment of virion-associated Brij 98 rafts in retroviral replication and illustrate the importance of the raft-promoting property of virion-associated cholesterol in HIV-1 replication.

Natural history of hepatitis B in perinatally infected carriers

Objectives: To establish natural seroconversion rates and incidence of hepatic pathology in perinatally infected hepatitis B carriers.

Methods: Seventy three perinatally infected hepatitis B carriers identified through maternal screening were evaluated. Fifty three were born to parents from the Indian subcontinent, nine were Oriental, six were Afro-Caribbean, and five were white. Median follow up was 10.24 (range 2.02–20.16) years.

Results: Only three of the children followed up had cleared hepatitis B surface antigen during this period, and 30% of the children had seroconverted to anti-HBe. Seroconversions to anti-HBe were observed in Asian (18/50) and white (4/5) children, but not in Oriental or Afro-Caribbean children. More girls (40%) than boys (23%) had seroconverted, but the difference was not significant. All children were asymptomatic with normal physical examination, growth, and development. Almost half (48%) of the hepatitis B e antigen (HBeAg) positive children had normal hepatic transaminases and liver function. Thirty five liver biopsies were performed in children with active virus replication (HBeAg or hepatitis B virus DNA positive) who were being considered for antiviral treatment as part of a clinical trial and were scored using the Ishak method. Two thirds (62%) of the children had mild hepatitis, 60% had mild fibrosis, and 18% had moderate to severe fibrosis. There was a weak correlation between histological evidence of hepatitis and hepatic transaminase activity, implying that biochemical monitoring of hepatic disease activity may be ineffective.

Conclusions: These asymptomatic hepatitis B virus carrier children remain infectious in the medium to long term with notable liver pathology. They should receive antiviral treatment to reduce infectivity and to prevent further progression of liver disease. Hepatic transaminases alone are not a reliable marker of liver pathology, and liver histology is essential before consideration for antiviral treatment.

Parasitized snails take the heat : a case of host manipulation?

Infection-induced changes in a host’s thermal physiology can represent (1) a generalized host response to infection, (2) a pathological side-effect of infection, or (3), provided the parasite’s development is temperature-dependent, a subtle case of host manipulation. This study investigates parasite-induced changes in the thermal biology of a first intermediate host infected by two castrating trematodes (genera Maritrema and Philophthalmus) using laboratory experiments and Weld surveys. The heat tolerance and temperatures selected by the snail, Zeacumantus subcarinatus, displayed alterations upon infection that differed between the two trematodes. Upon heating, snails infected by Maritrema sustained activity for longer durations than uninfected snails, followed by a more rapid recovery, and selected higher temperatures in a thermal gradient. These snails were also relatively abundant in high shore localities in the summer only, corresponding with seasonal elevated microhabitat temperatures. By contrast, Philophthalmus infected snails fell rapidly into a coma upon heating and did not display altered thermal preferences. The respective heat tolerance of each trematode corresponded with the thermal responses induced in the snail: Maritrema survived exposure to 40°C, while Philophthalmus was less heat tolerant. Although both trematodes infect the same tissues, Philophthalmus leads to a reduction in the host’s thermal tolerance, a response consistent with a pathological side effect. By contrast, Maritrema induces heat tolerance in the snail and withstood exposure to high heat. As the developmental rate and infectivity of Maritrema increase with temperature up to 25°C, one adaptive explanation for our findings is that Maritrema manipulates the snail’s thermal responses to exploit warm microhabitats.

Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1

We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1IIIB) produced in COS-7 cells transfected with HIV-1 proviral DNA and mutant, noninfectious HIV-1Lai particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNA(1,2Lys), tRNA(3Lys) (the putative primer for HIV-1 reverse transcriptase) and tRNA(Ile). Identification was accomplished by comparing the electrophoretic mobilities and RNase T1 digests with those of tRNA(3Lys) and tRNA(1,2Lys) purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNA(Lys) incorporation into HIV-1. However, only the wild-type virus contains tRNA(3Lys) tightly associated with the viral RNA genome. The identification of the tightly associated tRNA as tRNA(3Lys) is based upon an electrophoretic mobility identical to that of tRNA(3Lys) and the ability of this RNA to hybridize with a tRNA(3Lys)-specific DNA probe. In addition to the four wild-type tRNA species, the mutant HIV-1-like particle contains two tRNA(His) species and three tRNA-sized species that we have been unable to identify. Their absence in wild-type virus makes it unlikely that they are required for viral infectivity.

Incorporation of excess wild-type and mutant tRNA(3Lys) into human immunodeficiency virus type 1

Human immunodeficiency virus (HIV) particles produced in COS-7 cells transfected with HIV type 1 (HIV-1) proviral DNA contain 8 molecules of tRNA(3Lys) per 2 molecules of genomic RNA and 12 molecules of tRNA1,2Lys per 2 molecules of genomic RNA. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a human tRNA3Lys gene, there is a large increase in the amount of cytoplasmic tRNA3Lys per microgram of total cellular RNA, and the tRNA3Lys content in the virus increases from 8 to 17 molecules per 2 molecules of genomic RNA. However, the total number of tRNALys molecules per 2 molecules of genomic RNA remains constant at 20; i.e., the viral tRNA1,2Lys content decreases from 12 to 3 molecules per 2 molecules of genomic RNA. All detectable tRNA3Lys is aminoacylated in the cytoplasm of infected cells and deacylated in the virus. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a mutant amber suppressor tRNA3Lys gene (in which the anticodon is changed from TTT to CTA), there is also a large increase in the relative concentration of cytoplasmic tRNA3Lys, and the tRNA3Lys content in the virus increases from 8 to 15 molecules per 2 molecules of genomic RNA, with a decrease in viral tRNA1,2Lys from 12 to 5 molecules per 2 molecules of genomic RNA. Thus, the total number of molecules of tRNALys in the virion remains at 20. The alteration of the anticodon has little effect on the viral packaging of this mutant tRNA in spite of the fact that it no longer contains the modified base mcm 5s2U at position 34, and its ability to be aminoacylated is significantly impaired compared with that of wild-type tRNA3Lys. Viral particles which have incorporated either excess wild-type tRNA3Lys or mutant suppressor tRNA3Lys show no differences in viral infectivity compared with wild-type HIV-1.

The dimer initiation sequence stem-loop of human immunodeficiency virus type 1 is dispensable for viral replication in peripheral blood mononuclear cells

Human immunodeficiency virus type 1 (HIV-1) contains two copies of genomic RNA that are noncovalently linked via a palindrome sequence within the dimer initiation site (DIS) stem-loop. In contrast to the current paradigm that the DIS stem or stem-loop is critical for HIV-1 infectivity, which arose from studies using T-cell lines, we demonstrate here that HIV-1 mutants with deletions in the DIS stem-loop are replication competent in peripheral blood mononuclear cells (PBMCs). The DIS mutants contained either the wild-type (5′GCGCGC3′) or an arbitrary (5′ACGCGT3′) palindrome sequence in place of the 39-nucleotide DIS stem-loop (NLCGCGCG and NLACGCGT). These DIS mutants were replication defective in SupT1 cells, concurring with the current model in which DIS mutants are replication defective in T-cell lines. All of the HIV-1 DIS mutants were replication competent in PBMCs over a 40-day infection period and had retained their respective DIS mutations at 40 days postinfection. Although the stability of the virion RNA dimer was not affected by our DIS mutations, the RNA dimers exhibited a diffuse migration profile when compared to the wild type. No defect in protein processing of the Gag and GagProPol precursor proteins was found in the DIS mutants. Our data provide direct evidence that the DIS stem-loop is dispensable for viral replication in PBMCs and that the requirement of the DIS stem-loop in HIV-1 replication is cell type dependent.