The establishment of SARS CoV-2 spike-pseudotyped lentiviral (LV) systems has enabled the rapid identification of entry inhibitors and neutralizing agents, alongside allowing for the study of this emerging pathogen in BSL-2 level facilities. While such frameworks recapitulate the cellular entry process in ACE2+ cells, they are largely unable to factor in supplemental contributions by other SARS CoV-2 genes. To address this, we performed an unbiased ORF screen and identified the nucleoprotein (N) as a potent enhancer of spike-pseudotyped LV particle infectivity. We further demonstrate that the spike protein is better enriched in virions when the particles are produced in the presence of N protein. This enrichment of spike renders LV particles more infectious as well as less vulnerable to the neutralizing effects of a human IgG-Fc fused ACE2 microbody. Importantly, this improvement in infectivity is observed with both wild-type spike protein as well as the D614G mutant. Our results hold important implications for the design and interpretation of similar LV pseudotyping-based studies.

HIV-1 employs a rich arsenal of viral factors throughout its life cycle and co-opts intracellular trafficking pathways. This exquisitely coordinated process requires precise manipulation of the host microenvironment, most often within defined subcellular compartments. The virus capitalizes on the host by modulating cell-surface proteins and cleverly exploiting nuclear import pathways for post entry events, among other key processes. Successful virus–cell interactions are indeed crucial in determining the extent of infection. By evolving defenses against host restriction factors, while simultaneously exploiting host dependency factors, the life cycle of HIV-1 presents a fascinating montage of an ongoing host–virus arms race. Herein, we provide an overview of how HIV-1 exploits native functions of the host cell and discuss recent findings that fundamentally change our understanding of the post-entry replication events.

SERINC5 restricts nef-defective HIV-1 by affecting early steps of the virus life cycle. Distant retroviruses with a wide host-range encode virulent factors in response to the challenge by SERINC5. Yet, the evolutionary origins of this anti-retroviral activity, its prevalence among the paralogs, and its ability to target retroviruses remain understudied. In agreement with previous studies, we find that four human SERINC paralogs inhibit nef-defective HIV-1, with SERINC2 being an exception. Here, we demonstrate that this lack of activity in human SERINC2 is associated with its post-whole genome duplication (WGD) divergence, as evidenced by the ability of pre-WGD orthologs from yeast, fly, and a post-WGD-proximate SERINC2 from coelacanth to inhibit the virus. Intriguingly, Nef is unable to counter coelacanth SERINC2, indicating that such activity was directed towards other retroviruses found in coelacanth (like foamy viruses). However, foamy-derived vectors are intrinsically resistant to the action of SERINC2, and we show that the foamy virus envelope confers this resistance by affecting its steady-state levels. Our study highlights an ancient origin of anti-retroviral activity in SERINCs and a hitherto unknown interaction with a foamy virus. Importance SERINC5 constitutes a critical barrier to the propagation of retroviruses as highlighted by parallel emergence of anti-SERINC5 activities among distant retroviral lineages. Therefore, understanding the origin and evolution of these host factors will provide key information about virus-host relationships that can be exploited for future drug development. Here we show that SERINC5-mediated nef-defective HIV-1 infection inhibition is evolutionarily conserved. SERINC2 from coelacanth restricts HIV-1 and it was functionally adapted to target foamy viruses. Our findings provide insights into the evolutionary origin of anti-retroviral activity in SERINC gene family and uncover the role of SERINCs in shaping the long-term conflicts between retroviruses and their hosts.

In addition to Nef, HIV-1 envelope glycoprotein has been shown to modulate SERINC5-mediated inhibition. Counterintuitively, Nef from the same isolates preserves the ability to prevent SERINC5 incorporation into virions, implying additional functions of the host protein.

The establishment of SARS CoV-2 spike-pseudotyped lentiviral (LV) systems has enabled the rapid identification of entry inhibitors and neutralizing agents, alongside allowing for the study of this emerging pathogen in BSL-2 level facilities. While such frameworks recapitulate the cellular entry process in ACE2+ cells, they are largely unable to factor in supplemental contributions by other SARS CoV-2 genes. To address this, we performed an unbiased ORF screen and identified the nucleoprotein (N) as a potent enhancer of spike-pseudotyped LV particle infectivity. We further demonstrate that this augmentation by N renders LV spike particles less vulnerable to the neutralizing effects of a human IgG-Fc fused ACE2 microbody. Biochemical analysis revealed that the spike protein is better enriched in virions when the particles are produced in the presence of SARS CoV-2 nucleoprotein. Importantly, this improvement in infectivity is achieved without a concomitant increase in sensitivity towards RBD binding-based neutralization. Our results hold important implications for the design and interpretation of similar LV pseudotyping-based studies.

9The SERINC gene family comprises of five paralogs in humans of which SERINC3 and 10 SERINC5 inhibit HIV-1 infectivity and are counteracted by Nef. The origin of this anti-retroviral 11 activity, its prevalence among the remaining human paralogs, and its ability to target 12 retroviruses remain largely unknown. Here we show that despite their early divergence, the 13 anti-retroviral activity is functionally conserved among four human SERINC paralogs with 14 SERINC2 being an exception. The lack of activity in human SERINC2 is associated with its 15 post-whole genome duplication (WGD) divergence, as evidenced by the ability of pre-WGD 16 orthologs from yeast, fly, and a post-WGD-proximate SERINC2 from coelacanth to inhibit nef-17 defective HIV-1. Intriguingly, potent retroviral factors from HIV-1 and MLV are not able to relieve 18 the SERINC2-mediated particle infectivity inhibition, indicating that such activity was directed 19 towards other retroviruses that are found in coelacanth (like foamy viruses). However, foamy-20 derived vectors are intrinsically resistant to the action of SERINC2, and we show that a foamy 21 virus envelope confers this resistance. Despite the presence of weak arms-race signatures, the 22 functional reciprocal adaptation among SERINC2 and SERINC5 and, in response, the 23 emergence of antagonizing ability in foamy virus appears to have resulted from a long-term 24 conflict with the host. 25 26 27 28 Introduction 29Viruses have been exploiting the host machinery for their persistence, and, in response, the 30 host has continued to evolve increasingly intricate antiviral defense strategies. As part of this 31 ongoing arms-race, while viruses have relied on acquisition and fusion of diverse genes (1), 32 the host-defense mechanism has been made possible by the functional divergence of gene 33 copies following duplication of genes as well as whole-genomes (2-7). Restriction factors being 34 at the forefront of this long-term conflict (8-10), show clear molecular signatures of arms-race 35 (11,12). In fact, the presence of these signatures has been proposed to be a hallmark of 36 restriction factors (8,13), and has been used as a screening strategy to identify potential 37 candidates (2,14). In contrast, the recently identified anti-retroviral host inhibitors SERINC5 and 38 SERINC3 display an uneventful evolutionary history (15). This is counterintuitive, because 39 distant retroviruses, with wide host-range, encode anti-SERINC5 virulent factors (16)(17)(18). We, 40Ramdas et al.therefore, sought to trace the origins of this antiretroviral activity and its relevance for retroviral 41 inhibition. Our analysis to comprehend the evolutionary origins of the antiretroviral activity in 42 SERINCs, identifies an active SERINC2 with a hitherto unknown interaction with a foamy virus. 44 Results 45Antiretroviral activity among human SERINC paralogs 46 Analysis of sequence similarity and gene structure conservation reveals that SERINC5 and 47 SERINC4 share a recent ancestry (Fig-S1). Similarly, SERINC3 and...

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