Repellents of the maize pathogen Ustilago maydis are involved in formation of hydrophobic aerial hyphae and in cellular attachment. These peptides, called Rep1-1 to Rep1-11, are encoded by the rep1 gene and result from cleavage of the precursor protein Rep1 during passage of the secretion pathway. Using green fluorescent protein as a reporter, we here show that rep1 is expressed in filaments and not in the yeast form of U. maydis. In situ hybridization localized rep1 mRNA in the apex of the filament, which correlates with the expected site of secretion of the repellents into the cell wall. We also produced a synthetic peptide, Rep1-1. This peptide reduced the water surface tension to as low as 36 mJ m ؊2 . In addition, it formed amyloid-like fibrils as was shown by negative staining, by thioflavin T fluorescence, and by x-ray diffraction. These fibrils were not soluble in SDS but could be dissociated with trifluoroacetic acid. The repellents in the hyphal cell wall had a similar solubility and also stained with thioflavin T, strongly indicating that they are present as amyloid fibrils. However, such fibrils could not be observed at the hyphal surface. This can be explained by the fact that the Rep1-1 filaments decrease in length at increasing concentrations. Taken together, we have identified the second class of fungal proteins that form functional amyloid-like filaments at the hyphal surface.Ustilago maydis is the causal agent of smut in Zea mays (maize) and Euchlaena mexicana (Mexican teosinte). A filamentous pathogenic dikaryon is formed upon fusion of compatible yeast-like sporidia. Differentiation in the plant leads to the formation of diploid teliospores, which undergo meiosis ultimately resulting in haploid sporidia (see Refs. 1-3).Fusion of haploid cells and development of an infectious dikaryon are controlled by the a and b mating type loci. The a locus regulates cell fusion through a pheromone-based recognition system (4), and the b locus controls post-fusion steps of pathogenic development, including hyphal growth. The latter locus encodes two unrelated homeodomain proteins, bE and bW, that form heterodimers when they are derived from different alleles (5, 6). This heterodimer regulates a number of genes, among which the rep1 gene (7-10). This gene is highly expressed resulting in 2.5% of the mRNA. It encodes a preproprotein that, after processing at KEX2 recognition sites, results in 11 secreted peptides with a high sequence similarity. These peptides are localized in the cell wall of filaments, in an SDSinsoluble, but trifluoroacetic acid-extractable form (10). They are involved in formation of hydrophobic aerial hyphae (10) and in hyphal attachment to hydrophobic surfaces (11); as such they have functionally replaced hydrophobins in U. maydis (11).Hydrophobins, which are not related to the repellents, fulfill a wide spectrum of functions in fungal development (12). They do so by forming an amphipathic protein film, which consists of amyloid-like fibrils (13-15). We here show that the repellent Rep1...

The mRNAs encoding the Rev and Env proteins of simian immunodeficiency virus (SIV) are unique because upstream translation start codons are present that may modulate the expression of these viral proteins. This is true for the regular mRNAs, but we also report novel mRNA splicing variants that encode up to five upstream AUG (uAUG) codons. Their influence on Rev and Env translation was measured by mutational inactivation in reporter constructs and in the SIVmac239 strain. An intricate regulatory mechanism was disclosed that allows the virus to express a balanced amount of these two proteins. This insight also allows the design of vector constructs that efficiently express these proteins.

A novel genetic approach for the control of virus replication was used for the design of a conditionally replicating human immunodeficiency virus (HIV) variant, HIV-rtTA. HIV-rtTA gene expression and virus replication are strictly dependent on the presence of a non-toxic effector molecule, doxycycline (dox), and thus can be turned on and off at will in a graded and reversible manner. The in vivo replication capacity, pathogenicity and genetic stability of this HIV-rtTA variant were evaluated in a humanized mouse model of haematopoiesis that harbours lymphoid and myeloid components of the human immune system (HIS). Infection of dox-fed BALB Rag/γc HIS (BRG-HIS) mice with HIV-rtTA led to the establishment of a productive infection without CD4+ T-cell depletion. The virus did not show any sign of escape from dox control for up to 10 weeks after the onset of infection. No reversion towards a functional Tat–transactivating responsive (TAR) RNA element axis was observed, confirming the genetic stability of the HIV-rtTA variant in vivo. These results demonstrate the proof of concept that HIV-rtTA replicates efficiently in vivo. HIV-rtTA is a promising tool for fundamental research to study virus–host interactions in vivo in a controlled fashion.

The mRNAs encoding the Rev and Env proteins of simian immunodeficiency virus (SIV) are unique because upstream translation start codons are present that may modulate the expression of these viral proteins. We previously reported the regulatory effect of a small upstream open reading frame (ORF) on Rev and Env translation. Here we study this mechanism in further detail by modulating the strength of the translation signals upstream of the open reading frames in subgenomic reporters. Furthermore, the effects of these mutations on SIV gene expression and viral replication are analyzed. An intricate regulatory mechanism is disclosed that allows the virus to express a balanced amount of these two proteins.

Human immunodeficiency virus type 1 (HIV-1) is classified into different phylogenetic subtypes, with subtype C representing more than half of the novel infections globally. However, there are relatively few subtype C envelopes available for study. We amplified 18 unique env genes from 13 patients who were infected with subtype C HIV-1 in six African countries and in Scotland to create replication-competent viruses. These envelopes are phylogenetically diverse across the subtype C spectrum, and have on average more N-linked glycosylation sites and slightly longer variable loops than previously described C envelopes. We found that CCR3 coreceptor usage is less prevalent in subtype C than in subtype B viruses, and these envelopes have varied sensitivity to neutralization. The subtype C chimeric viruses generated in this study will be useful for evaluating the breadth of neutralizing antibodies and other entry inhibitors.Subtype C human immunodeficiency virus type 1 (HIV-1) accounts for .50 % of all new infections worldwide, and is the most prevalent subtype in sub-Saharan Africa, South Asia and Brazil (UNAIDS/WHO, 2009). Although useful panels of subtype C primary isolates (Brown et al., 2005;Bures et al., 2002;Fernandez-Garcia et al., 2009) and pseudoviruses (Gray et al., 2006;Li et al., 2006b) have been developed, more need to be characterized due to the diversity of the envelope gene, env, within this subtype. For instance, the standard reference panel of subtype C envelopes (Envs) included isolates from only two African countries, Zambia and South Africa (Li et al., 2006b). Here, we analysed Envs from infections acquired in six African countries and from Scotland, UK. Our replicationcompetent viruses with new Envs represent a diverse range of contemporary subtype C isolates for which we have determined coreceptor usage and sensitivity to neutralizing antibodies.Plasma samples were obtained from 13 HIV-1 subtype C infected, treatment naive individuals diagnosed in Scotland through the National Surveillance System (Yirrell et al., 2004). The undisclosed patients' origins and viral loads are presented in Supplementary Table S1 (available in JGV Online). Nine samples were derived from individuals infected in Africa and four from individuals infected in Scotland. The subtype was determined by gag sequence typing. Viral envs (gp160 and gp120) were amplified directly from plasma viral RNA extracted with QIAamp Viral RNA Mini kit (Qiagen) using primers shown in Supplementary Table S2 (available in JGV Online) and Titan One Tube RT-PCR kit (Roche Applied Sciences).Env genes were cloned into pHXB2Denv (gp120) or the C2 cassette (gp160), which enables production of replicationcompetent chimeric viruses (McKeating et al., 1996;Zheng & Daniels, 2001). Therefore, unlike the previous panels of subtype C envs, those in this panel are uniquely able to spread infection. Viruses were generated by transfection of 293T cells and clones that yielded infectious progeny [¢1000 focus forming units (f.f.u.) ml 21 on NP2/CD4/ CCR5 or NP2...

Tat has a pivotal role in human and simian immunodeficiency virus (HIV and SIV) replication because it stimulates transcription by binding to the trans-activator response (TAR) element. In addition, several other Tat functions have been proposed. Most studies have focused on HIV-1 Tat and much less is known about SIV Tat. An SIVmac239 variant was constructed previously in which the Tat-TAR transcription mechanism is functionally replaced by the doxycycline-inducible Tet-On gene expression mechanism (SIV-rtTA). In this study, SIV-rtTA variants were used to analyse the functions of SIV Tat. It was shown that Tat-minus SIV-rtTA variants replicated efficiently in PM1 T-cells, ruling out an additional essential Tat function. Nevertheless, replication was suboptimal in other cells, and evolutionary pressure to repair Tat expression was documented. It was demonstrated that SIV-rtTA required Tat for optimal gene expression, despite the absence of the Tat-TAR axis. This Tat effect was lost upon replacement of the long terminal repeat promoter region by a non-related promoter. These results indicate that Tat can activate SIV transcription via TAR RNA and U3 DNA elements but has no other essential function in replication in cultured cells. The experiments were limited to cell lines and PBMCs, and did not exclude an accessory Tat function under specific conditions or in vivo.

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