Infection length and host environment influence on Plasmodium falciparum dry season reservoir.

Persistence of malaria parasites in asymptomatic hosts is crucial in areas of seasonally-interrupted transmission, where P. falciparum bridges wet seasons months apart. During the dry season, infected erythrocytes exhibit extended circulation with reduced cytoadherence, increasing the risk of splenic clearance of infected cells and hindering parasitaemia increase. However, what determines parasite persistence for long periods of time remains unknown. Here, we investigated whether seasonality affects plasma composition so that P. falciparum can detect and adjust to changing serological cues; or if alternatively, parasite infection length dictates clinical presentation and persistency. Data from Malian children exposed to alternating ~6-month wet and dry seasons show that plasma composition is unrelated to time of year in non-infected children, and that carrying P. falciparum only minimally affects plasma constitution in asymptomatic hosts. Parasites persisting in the blood of asymptomatic children from the dry into the ensuing wet season rarely if ever appeared to cause malaria in their hosts as seasons changed. In vitro culture in the presence of plasma collected in the dry or the wet seasons did not affect parasite development, replication or host-cell remodelling. The absence of a parasite-encoded sensing mechanism was further supported by the observation of similar features in P. falciparum persisting asymptomatically in the dry season and parasites in age- and sex-matched asymptomatic children in the wet season. Conversely, we show that P. falciparum clones transmitted early in the wet season had lower chance of surviving until the end of the following dry season, contrasting with a higher likelihood of survival of clones transmitted towards the end of the wet season, allowing for the re-initiation of transmission. We propose that the decreased virulence observed in persisting parasites during the dry season is not due to the parasites sensing ability, nor is it linked to a decreased capacity for parasite replication but rather a consequence decreased cytoadhesion associated with infection length.

A Conserved Acidic Residue in the C-Terminal Flexible Loop of HIV-1 Nef Contributes to the Activity of SERINC5 and CD4 Downregulation.

The host transmembrane protein SERINC5 is incorporated into retrovirus particles and inhibits HIV-1 infectivity. The lentiviral Nef protein counteracts SERINC5 by downregulating it from the cell surface and preventing its incorporation into virions. The ability of Nef to antagonize the host factor varies in magnitude between different HIV-1 isolates. After having identified a subtype H nef allele unable to promote HIV-1 infectivity in the presence of SERINC5, we investigated the molecular determinants responsible for the defective counteraction of the host factor. Chimeric molecules with a subtype C Nef highly active against SERINC5 were constructed to locate Nef residues crucial for the activity against SERINC5. An Asn at the base of the C-terminal loop of the defective nef allele was found in place of a highly conserved acidic residue (D/E 150). The conversion of Asn to Asp restored the ability of the defective Nef to downregulate SERINC5 and promote HIV-1 infectivity. The substitution was also found to be crucial for the ability of Nef to downregulate CD4, but not for Nef activities that do not rely on the internalization of receptors from the cell surface, suggesting a general implication in promoting clathrin-mediated endocytosis. Accordingly, bimolecular fluorescence complementation revealed that the conserved acidic residue contributes to the recruitment of AP2 by Nef. Altogether, our results confirm that Nef downregulates SERINC5 and CD4 by engaging a similar machinery and indicates that, in addition to the di-leucine motif, other residues in the C-terminal flexible loop are important for the ability of the protein to sustain clathrin-mediated endocytosis.

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells.

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.