Rhesus TRIM5α disrupts the HIV-1 capsid at the inter-hexamer interfaces

TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5αrh containing coiled-coil and SPRY domains (CC-SPRYrh). Concentration-dependent binding of CC-SPRYrh to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRYrh, but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5αrh on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction. © 2011 Zhao et al

Five Questions about Viral Trafficking in Neurons

One of the most exciting areas in biology is the nervous system and how it works. Viral infections of the nervous system have provided exceptional insight at many levels, from pathogenesis to basic biology. The nervous system has evolved rather complicated barriers that facilitate access to nutrients and contact with the outside world, but block entry of pathogens and toxins [1]. However, when these barriers are reduced for any number of reasons, nervous system infections are possible. When they occur, they can be devastating and, even with good antiviral drugs, difficult to manage. Viral infections can enter the brain via the blood (e.g., HIV, various encephalitis viruses) or by spread inside neurons from the body surface (e.g., rabies and alpha herpes viruses) [2,3]. In vertebrates, the nervous system comprises a peripheral collection of neurons (the peripheral nervous system, PNS) and a central set found in the brain and spinal cord (the central nervous system, CNS). While neurons are central players in neurobiology, it is important to realize that the majority of cells that comprise the nervous system are highly specialized, nonneuronal cells (e.g., different types of glial cells) [4]. Cells of the immune system also engage with and signal to the PNS to affect changes in the CNS [5]. We will focus on neurons, despite the other cellular complexity, because neurons provide direct avenues for viral infection. Recognition that viral infection follows nerve pathways enabled the development of viruses for neuronal circuit tracing [6–8]

Evidence for Restriction of Ancient Primate Gammaretroviruses by APOBEC3 but Not TRIM5α Proteins

Because of evolutionary pressures imposed through episodic colonization by retroviruses, many mammals express factors, such as TRIM5α and APOBEC3 proteins, that directly restrict retroviral replication. TRIM5 and APOBEC restriction factors are most often studied in the context of modern primate lentiviruses, but it is likely that ancient retroviruses imposed the selective pressure that is evident in primate TRIM5 and APOBEC3 genes. Moreover, these antiretroviral factors have been shown to act against a variety of retroviruses, including gammaretroviruses. Endogenous retroviruses can provide a ‘fossil record’ of extinct retroviruses and perhaps evidence of ancient TRIM5 and APOBEC3 antiviral activity. Here, we investigate whether TRIM5 and APOBEC3 proteins restricted the replication of two groups of gammaretroviruses that were endogenized in the past few million years. These endogenous retroviruses appear quite widespread in the genomes of old world primates but failed to colonize the human germline. Our analyses suggest that TRIM5α proteins did not pose a major barrier to the cross-species transmission of these two families of gammaretroviruses, and did not contribute to their extinction. However, we uncovered extensive evidence for inactivation of ancient gammaretroviruses through the action of APOBEC3 cytidine deaminases. Interestingly, the identities of the cytidine deaminases responsible for inactivation appear to have varied in both a virus and host species–dependent manner. Overall, sequence analyses and reconstitution of ancient retroviruses from remnants that have been preserved in the genomes of modern organisms offer the opportunity to probe and potentially explain the evolutionary history of host defenses against retroviruses

C. elegans Germ Cells Show Temperature and Age-Dependent Expression of Cer1, a Gypsy/Ty3-Related Retrotransposon

Virus-like particles (VLPs) have not been observed in Caenorhabditis germ cells, although nematode genomes contain low numbers of retrotransposon and retroviral sequences. We used electron microscopy to search for VLPs in various wild strains of Caenorhabditis, and observed very rare candidate VLPs in some strains, including the standard laboratory strain of C. elegans, N2. We identified the N2 VLPs as capsids produced by Cer1, a retrotransposon in the Gypsy/Ty3 family of retroviruses/retrotransposons. Cer1 expression is age and temperature dependent, with abundant expression at 15°C and no detectable expression at 25°C, explaining how VLPs escaped detection in previous studies. Similar age and temperature-dependent expression of Cer1 retrotransposons was observed for several other wild strains, indicating that these properties are common, if not integral, features of this retroelement. Retrotransposons, in contrast to DNA transposons, have a cytoplasmic stage in replication, and those that infect non-dividing cells must pass their genomic material through nuclear pores. In most C. elegans germ cells, nuclear pores are largely covered by germline-specific organelles called P granules. Our results suggest that Cer1 capsids target meiotic germ cells exiting pachytene, when free nuclear pores are added to the nuclear envelope and existing P granules begin to be removed. In pachytene germ cells, Cer1 capsids concentrate away from nuclei on a subset of microtubules that are exceptionally resistant to microtubule inhibitors; the capsids can aggregate these stable microtubules in older adults, which exhibit a temperature-dependent decrease in egg viability. When germ cells exit pachytene, the stable microtubules disappear and capsids redistribute close to nuclei that have P granule-free nuclear pores. This redistribution is microtubule dependent, suggesting that capsids that are released from stable microtubules transfer onto new, dynamic microtubules to track toward nuclei. These studies introduce C. elegans as a model to study the interplay between retroelements and germ cell biology

Characterisation of assembly and ubiquitylation by the RBCC motif of Trim5α

The post-entry restriction factor Trim5α blocks infection of retroviral pathogens shortly after the virus gains entry to the cell, preventing reverse transcription and integration into the host genome. Central to the mechanism of restriction is recognition of the lattice of capsid protein that forms the inner-shell of the retrovirus. To recognise this lattice, Trim5α has been shown to assemble into a large hexagonal array, complementary to the capsid lattice. Structures of the Trim5α coiled-coil region reveal an elongated anti-parallel dimer consistent with the edges of this array placing the Bbox domain at each end of the coiled-coil to facilitate assembly. To investigate the nature of this assembly we have designed and characterised a monomeric version of the TRIM RBCC motif with a truncated coiled-coil. Biophysical characterisation by SEC-MALLS, AUC, and SAXS demonstrate that this construct forms compact folded domain that assembles into a trimer that would support the formation of a hexagonal lattice. Furthermore, the RING domain and elements of the coiled-coil region are shown to contribute to assembly. Ubiquitylation assays demonstrate that this assembly increases ubiquitylation activity providing a link from recognition of the capsid lattice and assembly to the activation of innate immune signalling and restriction