Hemagglutinin (HA) of influenza A has been reported as the key protein in viral infection. Therefore, the density and the dynamic pattern of this protein in viral envelope will affect the virus to infect target cells. We used a lentiviral system to study the influenza A H1N1 viral infection. Herein we demonstrate that the influenza non-structural proteins (NS) significantly promote viral infection. By substituting NS gene segment from an H1N1 genome set of A/WSN/1933 with the NS segment isolated from another H1N1 substrain genome set, China246, we found that viral infection tropism was significantly altered. The reassortant H1N1 shows almost identical infectivity compared with its parental virus, A/WSN/1933, for the human epithelial cell line HOT, but shows only 1/100 infectivity of its parental virus when infecting the Madin-Darby canine kidney (MDCK) cell line. These results suggest that not only is NS important in the infectivity of human influenza virus, but that it may play a critical role in viral tropism, allowing the virus to mutate and spread to other species. (C) 2012 Elsevier Inc. All rights reserved.