The accessory protein Nef of human immunodeficiency virus type 1 (HIV-1) has long been known to enhance the infectivity of HIV-1 progeny virions. The multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 were recently identified as novel antiviral proteins that restrict HIV-1 infectivity. Nef enhances HIV-1 infectivity by removing SERINCs from the plasma membrane, which prevents their incorporation into progeny HIV-1 virions. To exploit this potent intrinsic antiretroviral factor for potential therapy development, it is critical to explore the determinants in SERINC5 that govern its downregulation by Nef and its restriction on HIV-1 infectivity. Here I report that the ability to inhibit HIV-1 infectivity is conserved among vertebrate SERINC5 proteins, whereas the sensitivity to downregulation by Nef is not. However, a Nef-resistant SERINC5 became Nef-sensitive when its intracellular loop 4 (ICL4) was replaced by that of Nef-sensitive human SERINC5. Conversely, human SERINC5 became resistant to Nef when its ICL4 was replaced by that of a Nef-resistant SERINC5. In general, ICL4 regions from SERINCs that exhibited resistance to a given Nef conferred resistance to the same Nef when transferred to a sensitive SERINC, and vice versa. I demonstrate that human SERINC5 can be modified to restrict HIV-1 infectivity even in the presence of Nef. Moreover, by generating chimeras between SERINC5 and SERINC2, which does not exhibit antiretroviral activity, I demonstrate that SERINC5’s inhibitory function, unlike the sensitivity to Nef, requires the participation of more than one region. Helix 4 and extracellular loop 5 (ECL5) of SERINC5 are both required for the potent restriction of HIV-1 infectivity. In contrast, a large amino-terminal portion of SERINC5 is not required for its antiretroviral activity of SERINC5. The determinants in ECL5 disperse throughout the loop. Furthermore, the ECL5 of SERINC5 is a hotspot region that determines the Env-dependent antiretroviral activity of SERINC5.

We recently identified the multipass transmembrane protein SERINC5 as an antiviral protein that can potently inhibit HIV-1 infectivity and is counteracted by HIV-1 Nef. We now report that the anti-HIV-1 activity, but not the sensitivity to Nef, is conserved among vertebrate SERINC5 proteins. However, a Nef-resistant SERINC5 became Nef sensitive when its intracellular loop 4 (ICL4) was replaced by that of Nef-sensitive human SERINC5. Conversely, human SERINC5 became resistant to Nef when its ICL4 was replaced by that of a Nef-resistant SERINC5. In general, ICL4 regions from SERINCs that exhibited resistance to a given Nef conferred resistance to the same Nef when transferred to a sensitive SERINC, and vice versa. Our results establish that human SERINC5 can be modified to restrict HIV-1 infectivity even in the presence of Nef.

Influenza A virus (IAV) lacks the enzyme for adding 5' caps to its RNAs and snatches the 5' ends of host capped RNAs to prime transcription. Neither the preference of the host RNA sequences snatched nor the effect of cap-snatching on host processes is completely defined. Previous studies of influenza cap-snatching used poly(A)-selected RNAs from infected cells or relied on annotated host genes to define the snatched host RNAs, and thus lack details on many noncoding host RNAs including snRNAs, snoRNAs, and promoter-associated capped small (cs)RNAs, which are made by "paused" Pol II during transcription initiation. In this study, we used a nonbiased technique, CapSeq, to identify host and viral-capped RNAs including nonpolyadenylated RNAs in the same samples, and investigated the substrate-product correlation between the host RNAs and the viral RNAs. We demonstrated that noncoding host RNAs, particularly U1 and U2, are the preferred cap-snatching source over mRNAs or pre-mRNAs. We also found that csRNAs are highly snatched by IAV. Because the functions of csRNAs remain mostly unknown, especially in somatic cells, our finding reveals that csRNAs at least play roles in the process of IAV infection. Our findings support a model where nascent RNAs including csRNAs are the preferred targets for cap-snatching by IAV and raise questions about how IAV might use snatching preferences to modulate host-mRNA splicing and transcription.