An siRNA Screen of Membrane Trafficking Genes Highlights Pathways Common to HIV-1 and M-PMV Virus Assembly and Release

The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV) in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.     

Insights into cellular factors that regulate HIV-1 replication in human cells

Retroviruses integrate into the host cell's chromosome. Accordingly, many aspects of the life cycle of retroviruses like HIV-1 are intimately linked to the functions of cellular proteins and RNAs. In this review, we discuss in brief recent genomewide screens for the identification of cellular proteins that assist HIV-1 replication in human cells. We also review findings for other cellular moieties that help or restrict the viral life cycle.     

Translational control of retroviruses

All replication-competent retroviruses contain three main reading frames, gag, pol and env, which are used for the synthesis of structural proteins, enzymes and envelope proteins respectively. Complex retroviruses, such as lentiviruses, also code for regulatory and accessory proteins that have essential roles in viral replication. The concerted expression of these genes ensures the efficient polypeptide production required for the assembly and release of new infectious progeny virions. Retroviral protein synthesis takes place in the cytoplasm and depends exclusively on the translational machinery of the host infected cell. Therefore, not surprisingly, retroviruses have developed RNA structures and strategies to promote robust and efficient expression of viral proteins in a competitive cellular environment.     

Avian retroviral RNA element promotes unspliced RNA accumulation in the cytoplasm.

All retroviruses need mechanisms for nucleocytoplasmic export of their unspliced RNA and for maintenance of this RNA in the cytoplasm, where it is either translated to produce Gag and Pol proteins or packaged into viral particles. The complex retroviruses encode Rev or Rex regulatory proteins, which interact with cis-acting viral sequences to promote cytoplasmic expression of incompletely spliced viral RNAs. Since the simple retroviruses do not encode regulatory proteins, we proposed that they might contain cis-acting sequences that could interact with cellular Rev-like proteins. To test this possibility, we initially looked for a cis-acting sequence in avian retroviruses that could substitute for Rev and the Rev response element in human immunodeficiency virus type 1 expression constructs. A cis-acting element in the 3' untranslated region of Rous sarcoma virus (RSV) RNA was found to promote Rev-independent expression of human immunodeficiency virus type 1 Gag proteins. This element was mapped between RSV nucleotides 8770 and 8925 and includes one copy of the direct repeat (DR) sequences flanking the RSV src gene; similar activity was observed for the upstream DR. To address the function of this element in RSV, both copies of the DR sequence were deleted. Subsequently, each DR sequence was inserted separately back into this deleted construct. While the viral construct lacking both DR sequences failed to replicate, constructs containing either the upstream or downstream DR replicated well. In the absence of both DRs, Gag protein levels were severely diminished and cytoplasmic levels of unspliced viral RNA were significantly reduced; replacement of either DR sequence led to normal levels of Gag protein and cytoplasmic unspliced RNA.     

Interactions between human endogenous and exogenous retroviruses

Retrovirus genes have become inserted into the human genome for more than one million years. These retroviruses are now inactivated due to mutation, such as deletions or nonsense mutations. After mutation, retroviruses eventually become fixed in the genome in the endogenous form and exist as traces of ancient viruses. These retroviruses are called human endogenous retroviruses (HERVs). HERVs cannot make fully active viruses, but a number of viral proteins (or even virus particles) are expressed under various conditions. By comparison with ERVs, some exogenous retroviruses are still infectious and cause serious diseases threatening human life. Recent studies have shown that some elements of HERVs are closely related to other exogenous retroviruses, including human immunodeficiency virus (HIV). This review will describe the regulation and interaction between HERVs and other active viral infections. In addition, we introduce the development of vaccines and therapeutic agents against these viral infections through the use of HERV elements.     

Spontaneous heteromerization of gammaretrovirus envelope proteins: a possible novel mechanism of retrovirus restriction

The env gene of gammaretroviruses encodes a glycoprotein conserved among diverse retroviruses, except for the domains involved in receptor binding. Here we show that pairs of gammaretrovirus envelope proteins (from Friend virus and GALV or xenotropic viruses) assemble into heteromers when coexpressed. This assembly results in a strong inhibition of infectivity. An unrelated envelope protein does not assemble in heteromers with the gammaretrovirus glycoproteins tested and does not affect their infectivity, demonstrating the specificity of the mechanism we describe. We propose that the numerous copies of endogenous retroviral env genes conserved within mammalian genomes act as restriction factors against infectious retroviruses.